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.

Helios is a key transcriptional regulator of outer hair cell maturation.

The sensory cells that are responsible for hearing include the cochlear inner hair cells (IHCs) and outer hair cells (OHCs), with the OHCs being necessary for sound sensitivity and tuning1. Both cell types are thought to arise from common progenitors; however, our understanding of the factors that control the fate of IHCs and OHCs remains limited. Here we identify Ikzf2 (which encodes Helios) as an essential transcription factor in mice that is required for OHC functional maturation and hearing. Helios is expressed in postnatal mouse OHCs, and in the cello mouse model a point mutation in Ikzf2 causes early-onset sensorineural hearing loss. Ikzf2cello/cello OHCs have greatly reduced prestin-dependent electromotile activity, a hallmark of OHC functional maturation, and show reduced levels of crucial OHC-expressed genes such as Slc26a5 (which encodes prestin) and Ocm. Moreover, we show that ectopic expression of Ikzf2 in IHCs: induces the expression of OHC-specific genes; reduces the expression of canonical IHC genes; and confers electromotility to IHCs, demonstrating that Ikzf2 can partially shift the IHC transcriptome towards an OHC-like identity.

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.

Biophysical and morphological changes in inner hair cells and their efferent innervation in the ageing mouse cochlea.

KEY POINTS: Age-related hearing loss is a progressive hearing loss involving environmental and genetic factors, leading to a decrease in hearing sensitivity, threshold and speech discrimination. We compared age-related changes in inner hair cells (IHCs) between four mouse strains with different levels of progressive hearing loss. The surface area of apical coil IHCs (9-12 kHz cochlear region) decreases by about 30-40% with age. The number of BK channels progressively decreases with age in the IHCs from most mouse strains, but the basolateral membrane current profile remains unchanged. The mechanoelectrical transducer current is smaller in mice harbouring the hypomorphic Cdh23 allele Cdh23ahl (C57BL/6J; C57BL/6NTac), but not in Cdh23-repaired mice (C57BL/6NTacCdh23+ ), indicating that it could contribute to the different progression of hearing loss among mouse strains. The degree of efferent rewiring onto aged IHCs, most likely coming from the lateral olivocochlea fibres, was correlated with hearing loss in the different mouse strains. ABSTRACT: Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea, transducing acoustic information into electrical signals that are relayed to the afferent neurons. Functional changes in IHCs are a potential cause of age-related hearing loss. Here, we have investigated the functional characteristics of IHCs from early-onset hearing loss mice harbouring the allele Cdh23ahl (C57BL/6J and C57BL/6NTac), from late-onset hearing loss mice (C3H/HeJ), and from mice corrected for the Cdh23ahl mutation (C57BL/6NTacCdh23+ ) with an intermediate hearing phenotype. There was no significant loss of IHCs in the 9-12 kHz cochlear region up to at least 15 months of age, but their surface area decreased progressively by 30-40% starting from ∼6 months of age. Although the size of the BK current decreased with age, IHCs retained a normal KCNQ4 current and resting membrane potential. These basolateral membrane changes were most severe for C57BL/6J and C57BL/6NTac, less so for C57BL/6NTacCdh23+ and minimal or absent in C3H/HeJ mice. We also found that lateral olivocochlear (LOC) efferent fibres re-form functional axon-somatic connections with aged IHCs, but this was seen only sporadically in C3H/HeJ mice. The efferent post-synaptic SK2 channels appear prior to the establishment of the efferent contacts, suggesting that IHCs may play a direct role in re-establishing the LOC-IHC synapses. Finally, we showed that the size of the mechanoelectrical transducer (MET) current from IHCs decreased significantly with age in mice harbouring the Cdh23ahl allele but not in C57BL/6NTacCdh23+ mice, indicating that the MET apparatus directly contributes to the progression of age-related hearing loss.

MET currents and otoacoustic emissions from mice with a detached tectorial membrane indicate the extracellular matrix regulates Ca2+ near stereocilia.

KEY POINTS: The aim was to determine whether detachment of the tectorial membrane (TM) from the organ of Corti in Tecta/Tectb-/- mice affects the biophysical properties of cochlear outer hair cells (OHCs). Tecta/Tectb-/- mice have highly elevated hearing thresholds, but OHCs mature normally. Mechanoelectrical transducer (MET) channel resting open probability (Po ) in mature OHC is ∼50% in endolymphatic [Ca2+ ], resulting in a large standing depolarizing MET current that would allow OHCs to act optimally as electromotile cochlear amplifiers. MET channel resting Po in vivo is also high in Tecta/Tectb-/- mice, indicating that the TM is unlikely to statically bias the hair bundles of OHCs. Distortion product otoacoustic emissions (DPOAEs), a readout of active, MET-dependent, non-linear cochlear amplification in OHCs, fail to exhibit long-lasting adaptation to repetitive stimulation in Tecta/Tectb-/- mice. We conclude that during prolonged, sound-induced stimulation of the cochlea the TM may determine the extracellular Ca2+ concentration near the OHC's MET channels. ABSTRACT: The tectorial membrane (TM) is an acellular structure of the cochlea that is attached to the stereociliary bundles of the outer hair cells (OHCs), electromotile cells that amplify motion of the cochlear partition and sharpen its frequency selectivity. Although the TM is essential for hearing, its role is still not fully understood. In Tecta/Tectb-/- double knockout mice, in which the TM is not coupled to the OHC stereocilia, hearing sensitivity is considerably reduced compared with that of wild-type animals. In vivo, the OHC receptor potentials, assessed using cochlear microphonics, are symmetrical in both wild-type and Tecta/Tectb-/- mice, indicating that the TM does not bias the hair bundle resting position. The functional maturation of hair cells is also unaffected in Tecta/Tectb-/- mice, and the resting open probability of the mechanoelectrical transducer (MET) channel reaches values of ∼50% when the hair bundles of mature OHCs are bathed in an endolymphatic-like Ca2+ concentration (40 µM) in vitro. The resultant large MET current depolarizes OHCs to near -40 mV, a value that would allow optimal activation of the motor protein prestin and normal cochlear amplification. Although the set point of the OHC receptor potential transfer function in vivo may therefore be determined primarily by endolymphatic Ca2+ concentration, repetitive acoustic stimulation fails to produce adaptation of MET-dependent otoacoustic emissions in vivo in the Tecta/Tectb-/- mice. Therefore, the TM is likely to contribute to the regulation of Ca2+ levels around the stereocilia, and thus adaptation of the OHC MET channel during prolonged sound stimulation.

Pathophysiological changes in inner hair cell ribbon synapses in the ageing mammalian cochlea.

KEY POINTS: Age-related hearing loss (ARHL) is associated with the loss of inner hair cell (IHC) ribbon synapses, lower hearing sensitivity and decreased ability to understand speech, especially in a noisy environment. Little is known about the age-related physiological and morphological changes that occur at ribbon synapses. We show that the differing degrees of ARHL in four selected mouse stains is correlated with the loss of ribbon synapses, being most severe for the strains C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ -Repaired and lowest for C3H/HeJ. Despite the loss of ribbon synapses with age, the volume of the remaining ribbons increased and the size and kinetics of Ca2+ -dependent exocytosis in IHCs was unaffected, indicating the presence of a previously unknown degree of functional compensation at ribbon synapses. Although the age-related morphological changes at IHC ribbon synapses contribute to the different progression of ARHL, without the observed functional compensation hearing loss could be greater. ABSTRACT: Mammalian cochlear inner hair cells (IHCs) are specialized sensory receptors able to provide dynamic coding of sound signals. This ability is largely conferred by their ribbon synapses, which tether a large number of vesicles at the IHC's presynaptic active zones, allowing high rates of sustained synaptic transmission onto the afferent fibres. How the physiological and morphological properties of ribbon synapses change with age remains largely unknown. Here, we have investigated the biophysical and morphological properties of IHC ribbon synapses in the ageing cochlea (9-12 kHz region) of four mouse strains commonly used in hearing research: early-onset progressive hearing loss (C57BL/6J and C57BL/6NTac) and 'good hearing' strains (C57BL/6NTacCdh23+ and C3H/HeJ). We found that with age, both modiolar and pillar sides of the IHC exhibited a loss of ribbons, but there was an increased volume of those that remained. These morphological changes, which only occurred after 6 months of age, were correlated with the level of hearing loss in the different mouse strains, being most severe for C57BL/6NTac and C57BL/6J, less so for C57BL/6NTacCdh23+ and absent for C3H/HeJ strains. Despite the age-related reduction in ribbon number in three of the four strains, the size and kinetics of Ca2+ -dependent exocytosis, as well as the replenishment of synaptic vesicles, in IHCs was not affected. The degree of vesicle release at the fewer, but larger, individual remaining ribbon synapses colocalized with the post-synaptic afferent terminals is likely to increase, indicating the presence of a previously unknown degree of functional compensation in the ageing mouse cochlea.

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.

Age-related changes in the biophysical and morphological characteristics of mouse cochlear outer hair cells.

KEY POINTS: Age-related hearing loss (ARHL) is a very heterogeneous disease, resulting from cellular senescence, genetic predisposition and environmental factors (e.g. noise exposure). Currently, we know very little about age-related changes occurring in the auditory sensory cells, including those associated with the outer hair cells (OHCs). Using different mouse strains, we show that OHCs undergo several morphological and biophysical changes in the ageing cochlea. Ageing OHCs also exhibited the progressive loss of afferent and efferent synapses. We also provide evidence that the size of the mechanoelectrical transducer current is reduced in ageing OHCs, highlighting its possible contribution in cochlear ageing. ABSTRACT: Outer hair cells (OHCs) are electromotile sensory receptors that provide sound amplification within the mammalian cochlea. Although OHCs appear susceptible to ageing, the progression of the pathophysiological changes in these cells is still poorly understood. By using mouse strains with a different progression of hearing loss (C57BL/6J, C57BL/6NTac, C57BL/6NTacCdh23+ , C3H/HeJ), we have identified morphological, physiological and molecular changes in ageing OHCs (9-12 kHz cochlear region). We show that by 6 months of age, OHCs from all strains underwent a reduction in surface area, which was not a sign of degeneration. Although the ageing OHCs retained a normal basolateral membrane protein profile, they showed a reduction in the size of the K+ current and non-linear capacitance, a readout of prestin-dependent electromotility. Despite these changes, OHCs have a normal Vm and retain the ability to amplify sound, as distortion product otoacoustic emission thresholds were not affected in aged, good-hearing mice (C3H/HeJ, C57BL/6NTacCdh23+ ). The loss of afferent synapses was present in all strains at 15 months. The number of efferent synapses per OHCs, defined as postsynaptic SK2 puncta, was reduced in aged OHCs of all strains apart from C3H mice. Several of the identified changes occurred in aged OHCs from all mouse strains, thus representing a general trait in the pathophysiological progression of age-related hearing loss, possibly aimed at preserving functionality. We have also shown that the mechanoelectrical transduction (MET) current from OHCs of mice harbouring the Cdh23ahl allele is reduced with age, highlighting the possibility that changes in the MET apparatus could play a role in cochlear ageing.

Gata3 is required for the functional maturation of inner hair cells and their innervation in the mouse cochlea.

KEY POINTS: The physiological maturation of auditory hair cells and their innervation requires precise temporal and spatial control of cell differentiation. The transcription factor gata3 is essential for the earliest stages of auditory system development and for survival and synaptogenesis in auditory sensory afferent neurons. We show that during postnatal development in the mouse inner ear gata3 is required for the biophysical maturation, growth and innervation of inner hair cells; in contrast, it is required only for the survival of outer hair cells. Loss of gata3 in inner hair cells causes progressive hearing loss and accounts for at least some of the deafness associated with the human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. The results show that gata3 is critical for later stages of mammalian auditory system development where it plays distinct, complementary roles in the coordinated maturation of sensory hair cells and their innervation. ABSTRACT: The zinc finger transcription factor gata3 regulates inner ear development from the formation of the embryonic otic placode. Throughout development, gata3 is expressed dynamically in all the major cochlear cell types. Its role in afferent formation is well established but its possible involvement in hair cell maturation remains unknown. Here, we find that in heterozygous gata3 null mice (gata3+/- ) outer hair cells (OHCs) differentiate normally but their numbers are significantly lower. In contrast, inner hair cells (IHCs) survive normally but they fail to acquire adult basolateral membrane currents, retain pre-hearing current and efferent innervation profiles and have fewer ribbon synapses. Targeted deletion of gata3 driven by otoferlin-cre recombinase (gata3fl/fl otof-cre+/- ) in IHCs does not affect OHCs or the number of IHC afferent synapses but it leads to a failure in IHC maturation comparable to that observed in gata3+/- mice. Auditory brainstem responses in gata3fl/fl otof-cre+/- mice reveal progressive hearing loss that becomes profound by 6-7 months, whilst distortion product otoacoustic emissions are no different to control animals up to this age. Our results, alongside existing data, indicate that gata3 has specific, complementary functions in different cell types during inner ear development and that its continued expression in the sensory epithelium orchestrates critical aspects of physiological development and neural connectivity. Furthermore, our work indicates that hearing loss in human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome arises from functional deficits in IHCs as well as loss of function from OHCs and both afferent and efferent neurons.

The Coupling between Ca2+ Channels and the Exocytotic Ca2+ Sensor at Hair Cell Ribbon Synapses Varies Tonotopically along the Mature Cochlea.

The cochlea processes auditory signals over a wide range of frequencies and intensities. However, the transfer characteristics at hair cell ribbon synapses are still poorly understood at different frequency locations along the cochlea. Using recordings from mature gerbils, we report here a surprisingly strong block of exocytosis by the slow Ca2+ buffer EGTA (10 mM) in basal hair cells tuned to high frequencies (∼30 kHz). In addition, using recordings from gerbil, mouse, and bullfrog auditory organs, we find that the spatial coupling between Ca2+ influx and exocytosis changes from nanodomain in low-frequency tuned hair cells (∼<2 kHz) to progressively more microdomain in high-frequency cells (∼>2 kHz). Hair cell synapses have thus developed remarkable frequency-dependent tuning of exocytosis: accurate low-latency encoding of onset and offset of sound intensity in the cochlea's base and submillisecond encoding of membrane receptor potential fluctuations in the apex for precise phase-locking to sound signals. We also found that synaptic vesicle pool recovery from depletion was sensitive to high concentrations of EGTA, suggesting that intracellular Ca2+ buffers play an important role in vesicle recruitment in both low- and high-frequency hair cells. In conclusion, our results indicate that microdomain coupling is important for exocytosis in high-frequency hair cells, suggesting a novel hypothesis for why these cells are more susceptible to sound-induced damage than low-frequency cells; high-frequency inner hair cells must have a low Ca2+ buffer capacity to sustain exocytosis, thus making them more prone to Ca2+-induced cytotoxicity.SIGNIFICANCE STATEMENT In the inner ear, sensory hair cells signal reception of sound. They do this by converting the sound-induced movement of their hair bundles present at the top of these cells, into an electrical current. This current depolarizes the hair cell and triggers the calcium-induced release of the neurotransmitter glutamate that activates the postsynaptic auditory fibers. The speed and precision of this process enables the brain to perceive the vital components of sound, such as frequency and intensity. We show that the coupling strength between calcium channels and the exocytosis calcium sensor at inner hair cell synapses changes along the mammalian cochlea such that the timing and/or intensity of sound is encoded with high precision.

Connexin-Mediated Signaling in Nonsensory Cells Is Crucial for the Development of Sensory Inner Hair Cells in the Mouse Cochlea.

Mutations in the genes encoding for gap junction proteins connexin 26 (Cx26) and connexin 30 (Cx30) have been linked to syndromic and nonsyndromic hearing loss in mice and humans. The release of ATP from connexin hemichannels in cochlear nonsensory cells has been proposed to be the main trigger for action potential activity in immature sensory inner hair cells (IHCs), which is crucial for the refinement of the developing auditory circuitry. Using connexin knock-out mice, we show that IHCs fire spontaneous action potentials even in the absence of ATP-dependent intercellular Ca2+ signaling in the nonsensory cells. However, this signaling from nonsensory cells was able to increase the intrinsic IHC firing frequency. We also found that connexin expression is key to IHC functional maturation. In Cx26 conditional knock-out mice (Cx26Sox10-Cre), the maturation of IHCs, which normally occurs at approximately postnatal day 12, was partially prevented. Although Cx30 has been shown not to be required for hearing in young adult mice, IHCs from Cx30 knock-out mice exhibited a comprehensive brake in their development, such that their basolateral membrane currents and synaptic machinery retain a prehearing phenotype. We propose that IHC functional differentiation into mature sensory receptors is initiated in the prehearing cochlea provided that the expression of either connexin reaches a threshold level. As such, connexins regulate one of the most crucial functional refinements in the mammalian cochlea, the disruption of which contributes to the deafness phenotype observed in mice and DFNB1 patients. SIGNIFICANCE STATEMENT: The correct development and function of the mammalian cochlea relies not only on the sensory hair cells, but also on the surrounding nonsensory cells. Although the nonsensory cells have been largely implicated in the general homeostasis in the mature cochlea, their involvement in the initial functional differentiation of the sensory inner hair cells is less clear. Using mutant mouse models for the most common form of congenital deafness in humans, which are knock-outs for the gap-junction channels connexin 26 and connexin 30 genes, we show that defects in nonsensory cells prevented the functional maturation of inner hair cells. In connexin knock-outs, inner hair cells remained stuck at a prehearing stage of development and, as such, are unable to process sound information.

Restoration of auditory evoked responses by human ES-cell-derived otic progenitors.

Deafness is a condition with a high prevalence worldwide, produced primarily by the loss of the sensory hair cells and their associated spiral ganglion neurons (SGNs). Of all the forms of deafness, auditory neuropathy is of particular concern. This condition, defined primarily by damage to the SGNs with relative preservation of the hair cells, is responsible for a substantial proportion of patients with hearing impairment. Although the loss of hair cells can be circumvented partially by a cochlear implant, no routine treatment is available for sensory neuron loss, as poor innervation limits the prospective performance of an implant. Using stem cells to recover the damaged sensory circuitry is a potential therapeutic strategy. Here we present a protocol to induce differentiation from human embryonic stem cells (hESCs) using signals involved in the initial specification of the otic placode. We obtained two types of otic progenitors able to differentiate in vitro into hair-cell-like cells and auditory neurons that display expected electrophysiological properties. Moreover, when transplanted into an auditory neuropathy model, otic neuroprogenitors engraft, differentiate and significantly improve auditory-evoked response thresholds. These results should stimulate further research into the development of a cell-based therapy for deafness.

Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells.

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1(Bth/Bth)) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca(2+) permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1(Bth/Bth) OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca(2+) dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1(Bth/Bth) OHCs, whereas raising the intracellular concentration of the Ca(2+) chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca(2+) permeability of the mutated MET channel indirectly causing the Ca(2+) sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca(2+) influx as a compensatory mechanism. SIGNIFICANCE STATEMENT: In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry.

Absence of Neuroplastin-65 Affects Synaptogenesis in Mouse Inner Hair Cells and Causes Profound Hearing Loss.

The Neuroplastin gene encodes two synapse-enriched protein isoforms, Np55 and Np65, which are transmembrane glycoproteins that regulate several cellular processes, including the genesis, maintenance, and plasticity of synapses. We found that an absence of Np65 causes early-onset sensorineural hearing loss and prevented the normal synaptogenesis in inner hair cells (IHCs) in the newly identified mouse mutant pitch. In wild-type mice, Np65 is strongly upregulated in the cochlea from around postnatal day 12 (P12), which corresponds to the onset of hearing. Np65 was specifically localized at the presynaptic region of IHCs. We found that the colocalization of presynaptic IHC ribbons and postsynaptic afferent terminals is greatly reduced in pitch mutants. Moreover, IHC exocytosis is also reduced with mutant mice showing lower rates of vesicle release. Np65 appears to have a nonessential role in vision. We propose that Np65, by regulating IHC synaptogenesis, is critical for auditory function in mammals. SIGNIFICANCE STATEMENT: In the mammalian cochlea, the sensory inner hair cells (IHCs) encode auditory information. They do this by converting sound wave-induced mechanical motion of their hair bundles into an electrical current. This current generates a receptor potential that controls release of glutamate neurotransmitter from their ribbon synapses onto the auditory afferent fiber. We show that the synapse-enriched protein Np65, encoded by the Neuroplastin gene, is localized at the IHC presynaptic region. In mutant mice, absence of Np65 causes early-onset sensorineural hearing loss and prevents normal neurotransmitter release in IHCs and colocalization of presynaptic ribbons with postsynaptic afferents. We identified Neuroplastin as a novel deafness gene required for ribbon synapse formation and function, which is critical for sound perception in mammals.

Absence of plastin 1 causes abnormal maintenance of hair cell stereocilia and a moderate form of hearing loss in mice.

Hearing relies on the mechanosensory inner and outer hair cells (OHCs) of the organ of Corti, which convert mechanical deflections of their actin-rich stereociliary bundles into electrochemical signals. Several actin-associated proteins are essential for stereocilia formation and maintenance, and their absence leads to deafness. One of the most abundant actin-bundling proteins of stereocilia is plastin 1, but its function has never been directly assessed. Here, we found that plastin 1 knock-out (Pls1 KO) mice have a moderate and progressive form of hearing loss across all frequencies. Auditory hair cells developed normally in Pls1 KO, but in young adult animals, the stereocilia of inner hair cells were reduced in width and length. The stereocilia of OHCs were comparatively less affected; however, they also showed signs of degeneration in ageing mice. The hair bundle stiffness and the acquisition of the electrophysiological properties of hair cells were unaffected by the absence of plastin 1, except for a significant change in the adaptation properties, but not the size of the mechanoelectrical transducer currents. These results show that in contrast to other actin-bundling proteins such as espin, harmonin or Eps8, plastin 1 is dispensable for the initial formation of stereocilia. However, the progressive hearing loss and morphological defects of hair cells in adult Pls1 KO mice point at a specific role for plastin 1 in the preservation of adult stereocilia and optimal hearing. Hence, mutations in the human PLS1 gene may be associated with relatively mild and progressive forms of hearing loss.

Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells.

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, Ca(2+) entry through the open MET channels causes adaptation, rapidly reducing their open probability and resetting their operating range. It remains uncertain whether such Ca(2+)-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 Ca(2+) reversal potential or their hair bundles were exposed to the in vivo endolymphatic Ca(2+) 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 Ca(2+) 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 Ca(2+), intracellular Ca(2+) buffering, and membrane potential, by their common effect on intracellular free Ca(2+).

Presynaptic maturation in auditory hair cells requires a critical period of sensory-independent spiking activity.

The development of neural circuits relies on spontaneous electrical activity that occurs during immature stages of development. In the developing mammalian auditory system, spontaneous calcium action potentials are generated by inner hair cells (IHCs), which form the primary sensory synapse. It remains unknown whether this electrical activity is required for the functional maturation of the auditory system. We found that sensory-independent electrical activity controls synaptic maturation in IHCs. We used a mouse model in which the potassium channel SK2 is normally overexpressed, but can be modulated in vivo using doxycycline. SK2 overexpression affected the frequency and duration of spontaneous action potentials, which prevented the development of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their morphology or general cell development. By manipulating the in vivo expression of SK2 channels, we identified the "critical period" during which spiking activity influences IHC synaptic maturation. Here we provide direct evidence that IHC development depends upon a specific temporal pattern of calcium spikes before sound-driven neuronal activity.

Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2.

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.

In vivo and in vitro biophysical properties of hair cells from the lateral line and inner ear of developing and adult zebrafish.

Hair cells detect and process sound and movement information, and transmit this with remarkable precision and efficiency to afferent neurons via specialized ribbon synapses. The zebrafish is emerging as a powerful model for genetic analysis of hair cell development and function both in vitro and in vivo. However, the full exploitation of the zebrafish is currently limited by the difficulty in obtaining systematic electrophysiological recordings from hair cells under physiological recording conditions. Thus, the biophysical properties of developing and adult zebrafish hair cells are largely unknown. We investigated potassium and calcium currents, voltage responses and synaptic activity in hair cells from the lateral line and inner ear in vivo and using near-physiological in vitro recordings. We found that the basolateral current profile of hair cells from the lateral line becomes more segregated with age, and that cells positioned in the centre of the neuromast show more mature characteristics and those towards the edge retain a more immature phenotype. The proportion of mature-like hair cells within a given neuromast increased with zebrafish development. Hair cells from the inner ear showed a developmental change in current profile between the juvenile and adult stages. In lateral line hair cells from juvenile zebrafish, exocytosis also became more efficient and required less calcium for vesicle fusion. In hair cells from mature zebrafish, the biophysical characteristics of ion channels and exocytosis resembled those of hair cells from other lower vertebrates and, to some extent, those in the immature mammalian vestibular and auditory systems. We show that although the zebrafish provides a suitable animal model for studies on hair cell physiology, it is advisable to consider that the age at which the majority of hair cells acquire a mature-type configuration is reached only in the juvenile lateral line and in the inner ear from >2 months after hatching.

A reduction in Ptprq associated with specific features of the deafness phenotype of the miR-96 mutant mouse diminuendo.

miR-96 is a microRNA, a non-coding RNA gene which regulates a wide array of downstream genes. The miR-96 mouse mutant diminuendo exhibits deafness and arrested hair cell functional and morphological differentiation. We have previously shown that several genes are markedly downregulated in the diminuendo organ of Corti; one of these is Ptprq, a gene known to be important for maturation and maintenance of hair cells. In order to study the contribution that downregulation of Ptprq makes to the diminuendo phenotype, we carried out microarrays, scanning electron microscopy and single hair cell electrophysiology to compare diminuendo mutants (heterozygous and homozygous) with mice homozygous for a functional null allele of Ptprq. In terms of both morphology and electrophysiology, the auditory phenotype of mice lacking Ptprq resembles that of diminuendo heterozygotes, while diminuendo homozygotes are more severely affected. A comparison of transcriptomes indicates there is a broad similarity between diminuendo homozygotes and Ptprq-null mice. The reduction in Ptprq observed in diminuendo mice appears to be a major contributor to the morphological, transcriptional and electrophysiological phenotype, but does not account for the complete diminuendo phenotype.