Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, of which there are two types organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains 5-15 thousand terminally differentiated hair cells, and their survival is essential for hearing as they do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. Machine learning can be used to automate the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, rat, guinea pig, pig, primate, and human cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 107,000 hair cells which have been identified and annotated as either inner or outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair-cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to give other hearing research groups the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

Charge and lipophilicity are required for effective block of the hair-cell mechano-electrical transducer channel by FM1-43 and its derivatives.

The styryl dye FM1-43 is widely used to study endocytosis but behaves as a permeant blocker of the mechano-electrical transducer (MET) channel in sensory hair cells, loading rapidly and specifically into the cytoplasm of hair cells in a MET channel-dependent manner. Patch clamp recordings of mouse outer hair cells (OHCs) were used to determine how a series of structural modifications of FM1-43 affect MET channel block. Fluorescence microscopy was used to assess how the modifications influence hair-cell loading in mouse cochlear cultures and zebrafish neuromasts. Cochlear cultures were also used to evaluate otoprotective potential of the modified FM1-43 derivatives. Structure-activity relationships reveal that the lipophilic tail and the cationic head group of FM1-43 are both required for MET channel block in mouse cochlear OHCs; neither moiety alone is sufficient. The extent of MET channel block is augmented by increasing the lipophilicity/bulkiness of the tail, by reducing the number of positive charges in the head group from two to one, or by increasing the distance between the two charged head groups. Loading assays with zebrafish neuromasts and mouse cochlear cultures are broadly in accordance with these observations but reveal a loss of hair-cell specific labelling with increasing lipophilicity. Although FM1-43 and many of its derivatives are generally cytotoxic when tested on cochlear cultures in the presence of an equimolar concentration of the ototoxic antibiotic gentamicin (5 µM), at a 10-fold lower concentration (0.5 µM), two of the derivatives protect OHCs from cell death caused by 48 h-exposure to 5 µM gentamicin.

Large-scale annotated dataset for cochlear hair cell detection and classification.

Our sense of hearing is mediated by cochlear hair cells, localized within the sensory epithelium called the organ of Corti. There are two types of hair cells in the cochlea, which are organized in one row of inner hair cells and three rows of outer hair cells. Each cochlea contains a few thousands of hair cells, and their survival is essential for our perception of sound because they are terminally differentiated and do not regenerate after insult. It is often desirable in hearing research to quantify the number of hair cells within cochlear samples, in both pathological conditions, and in response to treatment. However, the sheer number of cells along the cochlea makes manual quantification impractical. Machine learning can be used to overcome this challenge by automating the quantification process but requires a vast and diverse dataset for effective training. In this study, we present a large collection of annotated cochlear hair-cell datasets, labeled with commonly used hair-cell markers and imaged using various fluorescence microscopy techniques. The collection includes samples from mouse, human, pig and guinea pig cochlear tissue, from normal conditions and following in-vivo and in-vitro ototoxic drug application. The dataset includes over 90'000 hair cells, all of which have been manually identified and annotated as one of two cell types: inner hair cells and outer hair cells. This dataset is the result of a collaborative effort from multiple laboratories and has been carefully curated to represent a variety of imaging techniques. With suggested usage parameters and a well-described annotation procedure, this collection can facilitate the development of generalizable cochlear hair cell detection models or serve as a starting point for fine-tuning models for other analysis tasks. By providing this dataset, we aim to supply other groups within the hearing research community with the opportunity to develop their own tools with which to analyze cochlear imaging data more fully, accurately, and with greater ease.

A critical period of prehearing spontaneous Ca2+ spiking is required for hair-bundle maintenance in inner hair cells.

Sensory-independent Ca2+ spiking regulates the development of mammalian sensory systems. In the immature cochlea, inner hair cells (IHCs) fire spontaneous Ca2+ action potentials (APs) that are generated either intrinsically or by intercellular Ca2+ waves in the nonsensory cells. The extent to which either or both of these Ca2+ signalling mechansims are required for IHC maturation is unknown. We find that intrinsic Ca2+ APs in IHCs, but not those elicited by Ca2+ waves, regulate the maturation and maintenance of the stereociliary hair bundles. Using a mouse model in which the potassium channel Kir2.1 is reversibly overexpressed in IHCs (Kir2.1-OE), we find that IHC membrane hyperpolarization prevents IHCs from generating intrinsic Ca2+ APs but not APs induced by Ca2+ waves. Absence of intrinsic Ca2+ APs leads to the loss of mechanoelectrical transduction in IHCs prior to hearing onset due to progressive loss or fusion of stereocilia. RNA-sequencing data show that pathways involved in morphogenesis, actin filament-based processes, and Rho-GTPase signaling are upregulated in Kir2.1-OE mice. By manipulating in vivo expression of Kir2.1 channels, we identify a "critical time period" during which intrinsic Ca2+ APs in IHCs regulate hair-bundle function.

Preferential Cochleotoxicity of Cisplatin.

Cisplatin-induced ototoxicity in humans is more predominant in the cochlea than in the vestibule. Neither definite nor substantial vestibular dysfunction after cisplatin treatment has been consistently reported in the current literature. Inner ear hair cells seem to have intrinsic characteristics that make them susceptible to direct exposure to cisplatin. The existing literature suggests, however, that cisplatin might have different patterns of drug trafficking across the blood-labyrinth-barrier, or different degrees of cisplatin uptake to the hair cells in the cochlear and vestibular compartments. This review proposes an explanation for the preferential cochleotoxicity of cisplatin based on current evidence as well as the anatomy and physiology of the inner ear. The endocochlear potential, generated by the stria vascularis, acting as the driving force for hair cell mechanoelectrical transduction might also augment cisplatin entry into cochlear hair cells. Better understanding of the stria vascularis might shed new light on cochleotoxic mechanisms and inform the development of otoprotective interventions to moderate cisplatin associated ototoxicity.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC's mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin-Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Impact of sight and hearing loss in patients with Norrie disease: advantages of Dual Sensory clinics in patient care.

Norrie disease (ND) is a rare, X-linked condition of visual and auditory impairment, often presenting with additional neurological features and developmental delays of varying severity. While all affected patients are born blind, or lose their vision in infancy, progressive sensorineural hearing loss develops in the majority of cases and is typically detected in the second decade of life. A range of additional symptoms of ND, such as seizure disorders, typically appear from a young age, but it is difficult to predict the range of symptoms ND patients will experience. After growing up without vision, hearing loss represents the greatest worry for many patients with ND, as they may lose the ability to participate in previously enjoyed activities or to communicate with others. Dual sensory loss has a physical, psychosocial and financial impact on both patients with ND and their families. Routine monitoring of the condition is required in order to identify, treat and provide support for emerging health problems, leading to a large burden of medical appointments. Many patients need to travel long distances to meet with specialists, representing a further burden on time and finances. Additionally, the rare nature of dual sensory impairment in children means that few clinical environments are designed to meet their needs. Dual Sensory clinics are multidisciplinary environments designed for sensory-impaired children and have been suggested to alleviate the impact of diseases involving sensory loss such as ND. Here, we discuss the diagnosis, monitoring and management of ND and the impact it has on paediatric patients and their caregivers. We describe the potential for dual sensory clinics to reduce disease burden through providing an appropriate clinical environment, access to multiple clinical experts in one visit, and ease of monitoring for patients with ND.

Hair cell maturation is differentially regulated along the tonotopic axis of the mammalian cochlea.

KEY POINTS: Outer hair cells (OHCs) enhance the sensitivity and the frequency tuning of the mammalian cochlea. Similar to the primary sensory receptor, the inner hair cells (IHCs), the mature functional characteristics of OHCs are acquired before hearing onset. We found that OHCs, like IHCs, fire spontaneous Ca2+ -induced action potentials (APs) during immature stages of development, which are driven by CaV 1.3 Ca2+ channels. We also showed that the development of low- and high-frequency hair cells is differentially regulated during pre-hearing stages, with the former cells being more strongly dependent on experience-independent Ca2+ action potential activity. ABSTRACT: Sound amplification within the mammalian cochlea depends upon specialized hair cells, the outer hair cells (OHCs), which possess both sensory and motile capabilities. In various altricial rodents, OHCs become functionally competent from around postnatal day 7 (P7), before the primary sensory inner hair cells (IHCs), which become competent at about the onset of hearing (P12). The mechanisms responsible for the maturation of OHCs and their synaptic specialization remain poorly understood. We report that spontaneous Ca2+ activity in the immature cochlea, which is generated by CaV 1.3 Ca2+ channels, differentially regulates the maturation of hair cells along the cochlea. Under near-physiological recording conditions we found that, similar to IHCs, immature OHCs elicited spontaneous Ca2+ action potentials (APs), but only during the first few postnatal days. Genetic ablation of these APs in vivo, using CaV 1.3-/- mice, prevented the normal developmental acquisition of mature-like basolateral membrane currents in low-frequency (apical) hair cells, such as IK,n (carried by KCNQ4 channels), ISK2 and IACh (α9α10nAChRs) in OHCs and IK,n and IK,f (BK channels) in IHCs. Electromotility and prestin expression in OHCs were normal in CaV 1.3-/- mice. The maturation of high-frequency (basal) hair cells was also affected in CaV 1.3-/- mice, but to a much lesser extent than apical cells. However, a characteristic feature in CaV 1.3-/- mice was the reduced hair cell size irrespective of their cochlear location. We conclude that the development of low- and high-frequency hair cells is differentially regulated during development, with apical cells being more strongly dependent on experience-independent Ca2+ APs.

Gentamicin Affects the Bioenergetics of Isolated Mitochondria and Collapses the Mitochondrial Membrane Potential in Cochlear Sensory Hair Cells.

Aminoglycoside antibiotics are widely prescribed to treat a variety of serious bacterial infections. They are extremely useful clinical tools, but have adverse side effects such as oto- and nephrotoxicity. Once inside a cell they are thought to cause mitochondrial dysfunction, subsequently leading to apoptotic cell death due to an increase in reactive oxygen species (ROS) production. Here we present evidence of a direct effect of gentamicin (the most commonly prescribed aminoglycoside) on the respiratory activities of isolated rat liver and kidney mitochondria. We show that gentamicin stimulates state 4 and inhibits state 3u respiratory rates, thereby reducing the respiratory control ratio (RCR) whilst simultaneously causing a collapse of the mitochondrial membrane potential (MtMP). We propose that gentamicin behaves as an uncoupler of the electron transport chain (ETC) - a hypothesis supported by our evidence that it reduces the production of mitochondrial ROS (MtROS). We also show that gentamicin collapses the MtMP in the sensory hair cells (HCs) of organotypic mouse cochlear cultures.

ORC-13661 protects sensory hair cells from aminoglycoside and cisplatin ototoxicity.

Aminoglycoside (AG) antibiotics are widely used to prevent life-threatening infections, and cisplatin is used in the treatment of various cancers, but both are ototoxic and result in loss of sensory hair cells from the inner ear. ORC-13661 is a new drug that was derived from PROTO-1, a compound first identified as protective in a large-scale screen utilizing hair cells in the lateral line organs of zebrafish larvae. Here, we demonstrate, in zebrafish larvae and in mouse cochlear cultures, that ORC-13661 provides robust protection of hair cells against both ototoxins, the AGs and cisplatin. ORC-13661 also prevents both hearing loss in a dose-dependent manner in rats treated with amikacin and the loading of neomycin-Texas Red into lateral line hair cells. In addition, patch-clamp recordings in mouse cochlear cultures reveal that ORC-13661 is a high-affinity permeant blocker of the mechanoelectrical transducer (MET) channel in outer hair cells, suggesting that it may reduce the toxicity of AGs by directly competing for entry at the level of the MET channel and of cisplatin by a MET-dependent mechanism. ORC-13661 is therefore a promising and versatile protectant that reversibly blocks the hair cell MET channel and operates across multiple species and toxins.

Design, Synthesis, and Biological Evaluation of a New Series of Carvedilol Derivatives That Protect Sensory Hair Cells from Aminoglycoside-Induced Damage by Blocking the Mechanoelectrical Transducer Channel.

Aminoglycosides (AGs) are broad-spectrum antibiotics used for the treatment of serious bacterial infections but have use-limiting side effects including irreversible hearing loss. Here, we assessed the otoprotective profile of carvedilol in mouse cochlear cultures and in vivo zebrafish assays and investigated its mechanism of protection which, we found, may be mediated by a block of the hair cell's mechanoelectrical transducer (MET) channel, the major entry route for the AGs. To understand the full otoprotective potential of carvedilol, a series of 18 analogues were prepared and evaluated for their effect against AG-induced damage as well as their affinity for the MET channel. One derivative was found to confer greater protection than carvedilol itself in cochlear cultures and also to bind more tightly to the MET channel. At higher concentrations, both carvedilol and this derivative were toxic in cochlear cultures but not in zebrafish, suggesting a good therapeutic window under in vivo conditions.

Coordinated calcium signalling in cochlear sensory and non-sensory cells refines afferent innervation of outer hair cells.

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca2+ signals from sensory and non-sensory cells.

Aminoglycoside- and Cisplatin-Induced Ototoxicity: Mechanisms and Otoprotective Strategies.

Ototoxicity refers to damage of inner ear structures (i.e., the cochlea and vestibule) and their function (hearing and balance) following exposure to specific in-hospital medications (i.e., aminoglycoside antibiotics, platinum-based drugs), as well as a variety of environmental or occupational exposures (e.g., metals and solvents). This review provides a narrative derived from relevant papers describing factors contributing to (or increasing the risk of) aminoglycoside and cisplatin-induced ototoxicity. We also review current strategies to protect against ototoxicity induced by these indispensable pharmacotherapeutic treatments for life-threatening infections and solid tumors. We end by highlighting several interventional strategies that are currently in development, as well as the diverse challenges that still need to be overcome to prevent drug-induced hearing loss.

Mechanotransduction is required for establishing and maintaining mature inner hair cells and regulating efferent innervation.

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.

Generating inner ear organoids containing putative cochlear hair cells from human pluripotent stem cells.

In view of the prevalence of sensorineural hearing defects in an ageing population, the development of protocols to generate cochlear hair cells and their associated sensory neurons as tools to further our understanding of inner ear development are highly desirable. We report herein a robust protocol for the generation of both vestibular and cochlear hair cells from human pluripotent stem cells which represents an advance over currently available methods that have been reported to generate vestibular hair cells only. Generating otic organoids from human pluripotent stem cells using a three-dimensional culture system, we show formation of both types of sensory hair cells bearing stereociliary bundles with active mechano-sensory ion channels. These cells share many morphological characteristics with their in vivo counterparts during embryonic development of the cochlear and vestibular organs and moreover demonstrate electrophysiological activity detected through single-cell patch clamping. Collectively these data represent an advance in our ability to generate cells of an otic lineage and will be useful for building models of the sensory regions of the cochlea and vestibule.

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death.

Aminoglycoside antibiotics are used to treat life-threatening bacterial infections but can cause deafness due to hair cell death in the inner ear. Compounds have been described that protect zebrafish lateral line hair cells from aminoglycosides, but few are effective in the cochlea. As the aminoglycosides interact with several ion channels, including the mechanoelectrical transducer (MET) channels by which they can enter hair cells, we screened 160 ion-channel modulators, seeking compounds that protect cochlear outer hair cells (OHCs) from aminoglycoside-induced death in vitro. Using zebrafish, 72 compounds were identified that either reduced loading of the MET-channel blocker FM 1-43FX, decreased Texas red-conjugated neomycin labeling, or reduced neomycin-induced hair cell death. After testing these 72 compounds, and 6 structurally similar compounds that failed in zebrafish, 13 were found that protected against gentamicin-induced death of OHCs in mouse cochlear cultures, 6 of which are permeant blockers of the hair cell MET channel. None of these compounds abrogated aminoglycoside antibacterial efficacy. By selecting those without adverse effects at high concentrations, 5 emerged as leads for developing pharmaceutical otoprotectants to alleviate an increasing clinical problem.

TMC2 Modifies Permeation Properties of the Mechanoelectrical Transducer Channel in Early Postnatal Mouse Cochlear Outer Hair Cells.

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (~>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (~P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant.

d-Tubocurarine and Berbamine: Alkaloids That Are Permeant Blockers of the Hair Cell's Mechano-Electrical Transducer Channel and Protect from Aminoglycoside Toxicity.

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.

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI.

KEY POINTS: 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. ABSTRACT: 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 Ca(2+) -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 Ca(2+) -dependent adaptation.