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Type I spiral ganglion neurons (SGNs) convey sound information to the central auditory pathway by forming synapses with inner hair cells (IHCs) in the mammalian cochlea. The molecular mechanisms regulating the formation of the post-synaptic density (PSD) in the SGN afferent terminals are still unclear. Here, we demonstrate that brain-specific angiogenesis inhibitor 1 (BAI1) is required for the clustering of AMPA receptors GluR2-4 (glutamate receptors 2-4) at the PSD. Adult Bai1-deficient mice have functional IHCs but fail to transmit information to the SGNs, leading to highly raised hearing thresholds. Despite the almost complete absence of AMPA receptor subunits, the SGN fibers innervating the IHCs do not degenerate. Furthermore, we show that AMPA receptors are still expressed in the cochlea of Bai1-deficient mice, highlighting a role for BAI1 in trafficking or anchoring GluR2-4 to the PSDs. These findings identify molecular and functional mechanisms required for sound encoding at cochlear ribbon synapses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Spiral ligament fibrocytes of the cochlea play homoeostatic roles in hearing and their degeneration contributes to hearing loss. Culturing fibrocytes in vitro provides a way to evaluate their functional characteristics and study possible therapies for hearing loss. We investigated whether in vivo characteristics of fibrocytes could be recapitulated in vitro by modifying the culture substrates and carried out proof of concept studies for potential transplantation of culture cells into the inner ear. Fibrocytes cultured from 4 to 5-week old CD/1 mice were grown on 2D substrates coated with collagen I, II, V or IX and, after harvesting, onto or into 3D substrates (hydrogels) of collagen I alone or mixed collagen I and II at a 1:1 ratio. We also assessed magnetic nanoparticle (MNP) uptake. Cell counts, immunohistochemical and ultrastructural studies showed that fibrocytes grown on 2D substrates proliferated, formed both small spindle-shaped and large flat cells that avidly took up MNPs. Of the different collagen coatings, only collagen II had an effect, causing a reduced size of the larger cells. On hydrogels, the cells were plump/rounded with extended processes, resembling native cells. They formed networks over the surface and became incorporated into the gel. In all culture formats, the majority co-expressed caldesmon, aquaporin 1, S-100 and sodium potassium ATPase, indicating a mixed or uncharacterised phenotype. Time-course experiments showed a decrease to ∼50% of the starting population by 4d after seeding on collagen I hydrogels, but better survival (∼60%) was found on collagen I + II gels, whilst TEM revealed the presence of apoptotic cells. Cells grown within gels additionally showed necrosis. These results demonstrate that fibrocytes grown in 3D recapitulate in vivo morphology of native fibrocytes, but have poorer survival, compared with 2D. Therefore hydrogel cultures could be used to study fibrocyte function and might also offer avenues for cell-replacement therapies, but need more optimization for therapeutic use. Fibrocyte function could be modified using MNPs in combination, for example, with gene transfection., Competing Interests: Declaration of Competing Interest The authors of this manuscript declare that they have no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

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.

Mechanosensory transduction by vertebrate hair cells depends on a protein complex at the tips of shorter stereocilia associated with mechanoelectrical transduction channels activated by tip links in the hair bundle. In mammalian hair cells, this complex includes transmembrane channel-like protein subunit 1 (TMC1), lipoma HMGIC fusion partner-like 5 protein (LHFPL5) and protocadherin 15 (PCDH15), a lower-end component of the tip link. TMC1 interacts with LHFPL5 and PCDH15 but how the complex develops to maturity, and the relationships between these proteins, remains uncertain. Here we evaluate the spatiotemporal development of LHFPL5 distributions in mouse cochlear hair bundles by immunofluorescence and immunogold transmission electron microscopy, from postnatal day 0 (P0) through P21 in wild type and PCDH15-deficient mice. At P0, hair bundles contain many short microvilli-like processes which we term unranked stereocilia, and a subset of lengthening rows, adjacent to a kinocilium. LHFPL5 is distributed throughout the bundle, including on stereocilia tips and the kinocilium. At P3, 4-to-6 rows of ranked stereocilia are evident, total LHFPL5 expression peaks, and LHFPL5 is localised to ranked stereocilia tips of all rows and to lower shaft/ankle links. By P12, the bundle has a mature pattern with 3 ranked rows but virtually no unranked stereocilia or kinocilium; LHFPL5 expression has declined and become restricted to the tips of shorter stereocilia. Throughout development from P0, expression of LHFPL5 is greater overall on apical than basal bundles, but there is, on average, an equal amount of labelling per labelled tip. In P3 mice lacking PCDH15, LHFPL5 labelling is not at the tips but is primarily on unranked stereocilia and lower lateral links. These data show that LHFPL5 is already present in the MET apparatus at P0 but requires PCDH15 at P3 to remain there. Shaft/ankle link localisation suggests it interacts with link proteins other than PCDH15.

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm(2) from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss., (© 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse. We investigated the role of clarin-1 using various in vitro and in vivo approaches. We show by immunohistochemistry and patch-clamp recordings of Ca(2+) currents and membrane capacitance from inner hair cells that clarin-1 is not essential for formation or function of ribbon synapse. However, reduced cochlear microphonic potentials, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] loading, and transduction currents pointed to diminished cochlear hair bundle function in Clrn1(-/-) mice. Electron microscopy of cochlear hair cells revealed loss of some tall stereocilia and gaps in the v-shaped bundle, although tip links and staircase arrangement of stereocilia were not primarily affected by Clrn1(-/-) mutation. Human clarin-1 protein expressed in transfected mouse cochlear hair cells localized to the bundle; however, the pathogenic variant p.N48K failed to localize to the bundle. The mouse model generated to study the in vivo consequence of p.N48K in clarin-1 (Clrn1(N48K)) supports our in vitro and Clrn1(-/-) mouse data and the conclusion that CLRN1 is an essential hair bundle protein. Furthermore, the ear phenotype in the Clrn1(N48K) mouse suggests that it is a valuable model for ear disease in CLRN1(N48K), the most prevalent Usher syndrome III mutation in North America.

Spiral ligament fibrocytes function in cochlear homeostasis, maintaining the endocochlear potential by participating in potassium recycling, and fibrocyte degeneration contributes to hearing loss. Their superficial location makes them amenable to replacement by cellular transplantation. Fibrocyte cultures offer one source of transplantable cells, but determining what fibrocyte types they contain and what phenotype transplanted cells may adopt is problematic. Here, we use immunogold electron microscopy to assess the relative expression of markers in native fibrocytes of the CD/1 mouse spiral ligament. Caldesmon and aquaporin 1 are expressed more in type III fibrocytes than any other type. S-100 is strongly expressed in types I, II, and V fibrocytes, and α1Na,K-ATPase is expressed strongly only in types II and V. By combining caldesmon or aquaporin 1 with S-100 and α1Na,K-ATPase, a ratiometric analysis of immunogold density distinguishes all except type II and type V fibrocytes. Other putative markers (creatine kinase BB and connective tissue growth factor) did not provide additional useful analytical attributes. By labeling serial sections or by double or triple labeling with combinations of three antibodies, this technique could be used to distinguish all except type II and type V fibrocytes in culture or after cellular transplantation into the lateral wall.

Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy. Early pathological changes were detected in basal turn spiral ligament fibrocytes and spiral ganglion, but the latter was variable as both satellite cells and neurons were normal in some cochleae. Light microscopic counts in the spiral ligament of 20-week-old mice revealed that of the five main types (types I-V), only type V fibrocytes showed no reduction in numbers compared with 3-week-old animals, and type IV showed the greatest losses. However, all types of fibrocyte showed subtle damage when examined using electron microscopy, in the form of swollen mitochondria, as early as 2 weeks. The extent of mitochondrial damage showed a degree of correspondence with the light microscopic pattern of fibrocyte loss in that types III and IV fibrocytes had the most abnormal mitochondria and type V the least, especially at early stages. By 10-15 weeks, ultrastructural features of fibrocyte damage were similar to longer term changes reported in gerbils. Stria vascularis, spiral ganglion and hair cells showed few consistent early signs of damage but became increasingly affected, lagging behind the fibrocyte damage. Our data suggest that fibrocyte pathology may precede other presbycusic changes; breakdown of homeostatic mechanisms to which they contribute may cause the subsequent degeneration of the hair cells. Overall, there were many similarities to presbycusic changes in other rodents and humans. Therefore, the features of accelerated aging in this mouse make it a suitable model for rapidly assessing possible strategies to prevent or ameliorate presbycusic changes.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells., Competing Interests: The authors have declared that no competing interests exist.

The local expression of proteins involved in handling glutamate may be regulated by the number and activity of synapses in regions of glutamatergic innervation. The systematically varying innervation of inner hair cells (IHCs) of the cochlea provides a model to test this suggestion. IHCs are glutamatergic and form a single row along the cochlear spiral. Along this row the number of afferent fibers terminating on IHCs increases toward the base, reaching a peak and thereafter declining. The afferents are segregated so that higher spontaneous rate fibers terminate on the pillar-cell side of the IHC and lower rate fibers terminate on the modiolar side. Using immunofluorescence and postembedding immunogold labeling, we investigated the distributions of the glutamate-aspartate transporter (GLAST or excitatory amino acid transporter 1), vesicular glutamate transporter (VGLUT1), and the AMPA receptor glutamate receptor 4 (GluR4) along the spiral. Immunofluorescent labeling for GLAST in IHC supporting cells increased in intensity to a peak in the region of 6-9 mm from the apex. Immunogold labeling for GLAST was greater overall in these cells in the 10 mm region than in the 1 mm region and also on the pillar-cell side of the IHC compared with the modiolar side. Immunogold labeling for GluR4 was confined to synaptic sites, represented by puncta in immunofluorescence. The relative numbers of puncta changed with a gradient similar to that of GLAST labeling. VGLUT1 labeling occurred in IHCs but showed no clear cochleotopic gradient. These data suggest that both the density of innervation and the activity levels of glutamatergic synapses may be involved in modulating regional expression of GLAST.

The glutamate/aspartate transporter GLAST is known to occur in the plasma membrane of supporting cells and their glialike processes around the synaptic region of inner hair cells of the mammalian cochlea. Its function there is presumably to take up glutamate following the release of this putative amino acid neurotransmitter from the inner hair cells. In this study, we have investigated whether GLAST is also associated with the outer hair cells using postembedding immunogold labeling. This is interesting because it is uncertain whether the outer hair cells have a functional synapse at which glutamate may be released. However, earlier ultrastructural studies of the afferent synapses in outer hair cells in several mammalian species have shown features normally associated with synaptic activity. These observations are confirmed and extended here in guinea pig where these afferent synapses have presynaptic bodies, putative synaptic vesicles, and coated pits associated with them. Immunoreactivity for GLAST was found along the plasma membranes of Deiters' cells, especially around the synaptic region of the hair cell, on processes wrapped around approaching nerve fibers. Semiquantitative analysis of the distribution of immunogold labeling of Deiters' cells confirmed that it was densest in the region adjacent to the synapses. There was also more labeling in apical than in basal regions of the cochlea in three of the four animals examined, suggesting an association with the number of afferent synapses, which are more numerous in apical regions. Interestingly, labeling also occurred in other regions of the cell membrane away from the afferent terminals. This suggests that glutamate uptake is also required away from the immediate vicinity of synapses, perhaps as a consequence of glutamate dispersal resulting from the mechanical displacement of the cochlear partition during stimulation. Nonetheless, the particular association of GLAST with the synaptic region of the outer hair cell implies that the latter have active afferent synapses at which glutamate is released.

Transmembrane channel-like protein 1 (TMC1) is thought to form the ion-conducting pore of the mechanoelectrical transducer (MET) channel in auditory hair cells. Using single-channel analysis and ionic permeability measurements, we characterized six missense mutations in the purported pore region of mouse TMC1. All mutations reduced the Ca2+ permeability of the MET channel, triggering hair cell apoptosis and deafness. In addition, Tmc1 p.E520Q and Tmc1 p.D528N reduced channel conductance, whereas Tmc1 p.W554L and Tmc1 p.D569N lowered channel expression without affecting the conductance. Tmc1 p.M412K and Tmc1 p.T416K reduced only the Ca2+ permeability. The consequences of these mutations endorse TMC1 as the pore of the MET channel. The accessory subunits, LHFPL5 and TMIE, are thought to be involved in targeting TMC1 to the tips of the stereocilia. We found sufficient expression of TMC1 in outer hair cells of Lhfpl5 and Tmie knockout mice to determine the properties of the channels, which could still be gated by hair bundle displacement. Single-channel conductance was unaffected in Lhfpl5−/− but was reduced in Tmie−/−, implying TMIE very likely contributes to the pore. Both the working range and half-saturation point of the residual MET current in Lhfpl5−/− were substantially increased, suggesting that LHFPL5 is part of the mechanical coupling between the tip-link and the MET channel. Based on counts of numbers of stereocilia per bundle, we estimate that each PCDH15 and LHFPL5 monomer may contact two channels irrespective of location. [ABSTRACT FROM AUTHOR]

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. 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. 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 FROM AUTHOR]

Cochlear fibrocytes are a homeostatic supporting cell type embedded in the vascularized extracellular matrix of the spiral ligament, within the lateral wall. Here, they participate in the connective tissue syncytium that enables potassium recirculation into the scala media to take place and ensures development of the endolymphatic potential that helps drive current into hair cells during acoustic stimulation. They have also been implicated in inflammatory responses in the cochlea. Some fibrocytes interact closely with the capillaries of the vasculature in a way which suggests potential involvement, together with the stria vascularis , also in the blood-labyrinth barrier. Several lines of evidence suggests that pathology of the fibrocytes, along with other degenerative changes in this region, contribute to metabolic hearing loss (MHL) during aging that is becoming recognized as distinct from, and potentially a precursor for, sensorineural hearing loss (SNHL). This pathology may underlie a significant proportion of cases of presbycusis. Some evidence points also to an association between fibrocyte degeneration and Ménière's disease (MD). Fibrocytes are mesenchymal; this characteristic, and their location, make them amenable to potential cell therapy in the form of cell replacement or genetic modification to arrest the process of degeneration that leads to MHL. This review explores the properties and roles of this neglected cell type and suggests potential therapeutic approaches, such as cell transplantation or genetic engineering of fibrocytes, which could be used to prevent this form of presbycusis or provide a therapeutic avenue for MD. [ABSTRACT FROM AUTHOR]

Mechanoelectrical transducer (MET) currents were recorded from cochlear hair cells in mice with mutations of transmembrane channellike protein TMC1 to study the effects onMET channel properties. We characterized a Tmc1 mouse with a single-amino-acid mutation (D569N), homologous to a dominant human deafness mutation. Measurements were made in both Tmc2 wild-type and Tmc2 knockout mice. By 30 d, Tmc1 pD569N heterozygote mice were profoundly deaf, and there was substantial loss of outer hair cells (OHCs). MET current in OHCs of Tmc1 pD569N mutants developed over the first neonatal week to attain a maximum amplitude one-third the size of that in Tmc1 wild-type mice, similar at apex and base, and lacking the tonotopic size gradient seen in wild type. The MET-channel Ca2+ permeability was reduced 3-fold in Tmc1 pD569N homozygotes, intermediate deficits being seen in heterozygotes. Reduced Ca2+ permeability resembled that of the Tmc1 pM412K Beethoven mutant, a previously studied semidominant mouse mutation. The MET channel unitary conductance, assayed by single-channel recordings and by measurements of current noise, was unaffected in mutant apical OHCs. We show that, in contrast to the Tmc1 M412K mutant, there was reduced expression of the TMC1 D569N channel at the transduction site assessed by immunolabeling, despite the persistence of tip links. The reduction inMET channel Ca2+ permeability seen in both mutants may be the proximate cause of hair-cell apoptosis, but changes in bundle shape and protein expression in Tmc1 D569N suggest another role for TMC1 apart from forming the channel. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Presbycusis (age-related hearing loss) can result from various cochlear pathologies. We have studied the time course of degeneration in a mouse that shows accelerated presbycusis, the CD/1 mouse, as a possible model to investigate stem-cell strategies to prevent or ameliorate presbycusic changes. CD/1 mice from 0 to 72 weeks old were examined by light and electron microscopy. Early pathological changes were detected in basal turn spiral ligament fibrocytes and spiral ganglion, but the latter was variable as both satellite cells and neurons were normal in some cochleae. Light microscopic counts in the spiral ligament of 20-week-old mice revealed that of the five main types (types I-V), only type V fibrocytes showed no reduction in numbers compared with 3-week-old animals, and type IV showed the greatest losses. However, all types of fibrocyte showed subtle damage when examined using electron microscopy, in the form of swollen mitochondria, as early as 2 weeks. The extent of mitochondrial damage showed a degree of correspondence with the light microscopic pattern of fibrocyte loss in that types III and IV fibrocytes had the most abnormal mitochondria and type V the least, especially at early stages. By 10-15 weeks, ultrastructural features of fibrocyte damage were similar to longer term changes reported in gerbils. Stria vascularis, spiral ganglion and hair cells showed few consistent early signs of damage but became increasingly affected, lagging behind the fibrocyte damage. Our data suggest that fibrocyte pathology may precede other presbycusic changes; breakdown of homeostatic mechanisms to which they contribute may cause the subsequent degeneration of the hair cells. Overall, there were many similarities to presbycusic changes in other rodents and humans. Therefore, the features of accelerated aging in this mouse make it a suitable model for rapidly assessing possible strategies to prevent or ameliorate presbycusic changes. [ABSTRACT FROM AUTHOR]

Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca2+ dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca2+ sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca2+ dependence of immature spiking cells. However, the nature of the Ca2+ sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca2+ dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca2+ dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca2+ sensitivity of vesicle fusion at hair cell synapses. [ABSTRACT FROM AUTHOR]

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, β-actin, γ-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and β- and γ-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall. [ABSTRACT FROM AUTHOR]