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In the developing cochlea, just before the onset of hearing on postnatal day 12, the medial olivocochlear efferent axons in synaptic contact with the inner hair cells (IHCs) start withdrawing and new efferent synaptic connections are formed on the outer hair cells (OHCs), thereby progressing towards the adult pattern of medial olivocochlear efferent innervation. The synapses are inhibitory, calcium influx through the α9α10 nicotinic acetylcholine receptors (nAChRs) driving opening of calcium-dependent potassium channels. The nAChRs appear to function similarly in IHCs and OHCs, although with probable kinetic differences. Our aim was to assess their functional similarity in the neonatal mouse cochlea by making whole-cell recordings from both hair cell types between postnatal day 7 and 10 when nAChRs are expressed. ACh was applied to voltage-clamped hair cells by pressure-ejection from a pipette. The cells were dialysed with a Cs + -based solution designed to eliminate calcium-dependent potassium currents. There were differences in amplitude, voltage-sensitivity and reversal potential of the nAChR currents between IHCs and OHCs. There was also some indication that IHC nAChRs have slower activation and desensitization kinetics, although the relatively slow ACh application limited interpretation of this result. These differences, particularly concerning the reversal potential, might indicate the presence of different auxiliary protein subunits of the α9α10 receptor in neonatal IHCs and OHCs., Competing Interests: Declarations of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Inner ear hair cells assemble mechanosensitive hair bundles on their apical surface that transduce sounds and accelerations. Each hair bundle is comprised of ∼ 100 individual stereocilia that are arranged into rows of increasing height and width; their specific and precise architecture being necessary for mechanoelectrical transduction (MET). The actin cytoskeleton is fundamental to establishing this architecture, not only by forming the structural scaffold shaping each stereocilium, but also by composing rootlets and the cuticular plate that together provide a stable foundation supporting each stereocilium. In concert with the actin cytoskeleton, a large assortment of actin-binding proteins (ABPs) function to cross-link actin filaments into specific topologies, as well as control actin filament growth, severing, and capping. These processes are individually critical for sensory transduction and are all disrupted in hereditary forms of human hearing loss. In this review, we provide an overview of actin-based structures in the hair bundle and the molecules contributing to their assembly and functional properties. We also highlight recent advances in mechanisms driving stereocilia elongation and how these processes are tuned by MET., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Of all the human body's sensory systems, the auditory system is perhaps its most intricate. Hearing loss can result from even modest damage or cell death in the inner ear, and is the most common form of sensory loss. Human hearing is made possible by the sensory epithelium, the lateral wall, and auditory nerves. The most prominent functional cells in the sensory epithelium are outer hair cells (OHCs), inner hair cells (IHCs), and supporting cells. Different sound frequencies are processed by OHCs and IHCs in different cochlear regions, with those in the apex responsible for low frequencies and those in the basal region responsible for high frequencies. Hair cells can be damaged or destroyed by loud noise, aging process, genetic mutations, ototoxicity, infection, and illness. As such, they are a primary target for treating sensorineural hearing loss. Other areas known to affect hearing include spiral ganglion neurons (SGNs) in the auditory nerve. Age-related degradation of HCs and SGNs can also cause hearing loss. The aim of this review is to introduce the roles of mitochondria in human auditory system and the inner ear's main cell types and cellular functions, before going on to detail the likely health benefits of iPSC technology. We posit that patient-specific iPSCs with mitochondrial gene mutations will be an important aspect of regenerative medicine and will lead to significant progress in the treatment of SNHL., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Inner ear hair cells are mechanosensitive cells responsible for sensing and transmitting signals to the brain to be interpreted as sound or head position/movement. The zinc-finger protein, gfi1, is expressed in differentiating neurons and inner ear hair cells. Gfi1 deficiency leads to a massive loss of cochlear hair cells in mice. However, the mechanism remains unclear. To develop an effective molecular therapy for hearing loss, it is critical to first understand the relationship between gfi1 and hair cell development. We demonstrated in the zebrafish model that gfi1.2 was initially expressed in the inner ear at 14 h post-fertilization (hpf), preceding the expression of gfi1.1 at 19 hpf. In the morpholino-mediated gfi1.2 knockdown mutants, hair cells reduced in number without altering the expression of pax2a, dlx3b, atho1a and pou4f3, the markers for otic patterning and specification. There was a down-regulation of the pro-neuronal genes, ngn1 and atoh1b in the context of gfi1.2 knockdown, which was rescued by the exogenous gfi1.2. We also found that gfi1.2 may regulate ngn1 expression by suppressing id2a. Our results suggested that knockdown of gfi1.2 may lead to deafness through promoting cells proliferation in the pro-sensory region and interrupting cell differentiation., (Copyright © 2020. Published by Elsevier B.V.)

Proper functioning of the auditory nerve is of critical importance for auditory rehabilitation by cochlear implants. Here we used the Cldn14 -/- mouse to study in detail the effects of Claudin 14 loss on auditory synapses and the auditory nerve. Mutations in the tight junction protein Claudin 14 cause autosomal recessive non-syndromic hearing loss (DFNB29) in humans and mice, due to extensive degeneration of outer and inner hair cells. Here we show that massive inner hair cell loss in Cldn14 -/- mice starts after the third postnatal week. Immunohistochemical analysis, using presynaptic Ribeye and postsynaptic GluR2 or PSD 95 as markers, revealed the degeneration of full ribbon synapses in inner hair cells from apical cochlear regions already at postnatal day 12 (P12). At P20, significant reduction in number of ribbon synapses has been observed for all cochlear regions and the loss of synaptic ribbons becomes even more prominent in residual inner hair cells from middle and apical cochlear regions at P45, which by then lost more than 40% of all ribbon synapses. In contrast to excessive noise exposure, loss of Claudin 14 does not cause an increase in "orphan" ribbons with no postsynaptic counterpart due to a reduction of postsynaptic structures. Hair cell loss in Cldn14 -/- mice is associated with regression of peripheral auditory nerve processes, especially of outer radial fibers, which normally innervate the outer hair cells. The number of spiral ganglion neurons per area, however, was unchanged between the genotypes. Different effects were observed in the cochlear nucleus complex (CNC), the central projection area of the auditory nerve. While the dorsal cochlear nucleus (DCN) showed a significant 19.7% volume reduction, VGLUT-1 input was reduced by 34.4% in the ventral cochlear nucleus (VCN) but not in the DCN of Cldn14 -/- mice. Taken together, massive inner hair cell loss starts after the third postnatal week in Cldn14 -/- mice, but is preceded by the loss of ribbon synapses, which may be a first sign of an ongoing degeneration process in otherwise morphologically inconspicuously inner hair cells. In addition to the regression of peripheral nerve processes, reduced levels of VGLUT-1 in the VCN of Cldn14 -/- mice suggests that Claudin 14 loss does not only cause hair cell loss but also affects peripheral and central connectivity of the auditory nerve., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In different animal models, auditory nerve fibers display variation in spontaneous activity and response threshold. Functional and structural differences among inner hair cell ribbon synapses are believed to contribute to this variation. The relative volumes of synaptic proteins at individual synapses might be one such difference. This idea is based on the observation of opposing volume gradients of the presynaptic ribbons and associated postsynaptic glutamate receptor patches in mice along the pillar modiolar axis of the inner hair cell, the same axis along which fibers were shown to vary in their physiological properties. However, it is unclear whether these opposing gradients are expressed consistently across animal models. In addition, such volume gradients observed for separate populations of presynaptic ribbons and postsynaptic glutamate receptor patches suggest different relative volumes of these synaptic structures at individual synapses; however, these differences have not been examined in mice. Furthermore, it is unclear whether such gradients are limited to these synaptic proteins. Therefore, we analyzed organs of Corti isolated from CBA/CaJ, C57BL/6, and FVB/NJ mice using immunofluorescence, confocal microscopy, and quantitative image analysis. We find consistent expression of presynaptic volume gradients across strains of mice and inconsistent expression of postsynaptic volume gradients. We find differences in the relative volume of synaptic proteins, but these are different between CBA/CaJ mice, and C57BL/6 and FVB/NJ mice. We find similar results in C57BL/6 and FVB/NJ mice when using other postsynaptic density proteins (Shank1, Homer, and PSD95). These results have implications for the mechanisms by which volumes of synaptic proteins contribute to variations in the physiology of individual auditory nerve fibers and their vulnerability to excitotoxicity., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The retinoblastoma family of pocket proteins (pRBs), composed of Rb1, p107, and p130 are negative regulators of cell-cycle progression. The deletion of any individual pRB in the auditory system triggers hair cells' (HCs) and supporting cells' (SCs) proliferation to different extents. Nevertheless, accessing their combined role in the inner ear through conditional or complete knockout methods is limited by the early mortality of the triple knockout. In quiescent cells, hyperphosphorylation and inactivation of the pRBs are maintained through the activity of the Cyclin-D1-cdk4/6 complex. Cyclin D1 (CycD1) is expressed in the embryonic and neonatal inner ear. In the mature organ of Corti (OC), CycD1 expression is significantly downregulated, paralleling the OC mitotic quiescence. Earlier studies showed that CycD1 overexpression leads to cell-cycle reactivation in cultures of inner ear explants. Here, we characterize a Cre-activated, Doxycycline (Dox)-controlled, conditional CycD1 overexpression model, which when bred to a tetracycline-controlled transcriptional activator and the Atoh1-cre mouse lines, allow for transient CycD1 overexpression and pRBs' downregulation in the inner ear in a reversible fashion. Analyses of postnatal mice's inner ears at various time points revealed the presence of supernumerary cells throughout the length of the cochlea and in the vestibular end-organs. Notably, most supernumerary cells were observed in the inner hair cells' (IHCs) region, expressed myosin VIIa (M7a), and showed no signs of apoptosis at any of the time points analyzed. Auditory and vestibular phenotypes were similar between the different genotypes and treatment groups. The fact that no significant differences were observed in auditory and vestibular function supports the notion that the supernumerary cells detected in the adult mice cochlea and macular end-organs may not impair auditory functions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF's effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Advances in microelectromechanical systems (MEMS) technologies are enhancing the development of intracochlear delivery devices for the treatment of hearing loss with emerging pharmacological therapies. Direct intracochlear delivery addresses the limitations of systemic and intratympanic delivery. However, optimization of delivery parameters for these devices requires pharmacokinetic assessment of the spatiotemporal drug distribution inside the cochlea. Robust methods of measuring drug concentration in the perilymph have been developed, but lack spatial resolution along the tonotopic axis or require complex physiological measurements. Here we describe an approach for quantifying distribution of fluorescent drug-surrogate probe along the cochlea's sensory epithelium with high spatial resolution enabled by confocal fluorescence imaging. Fluorescence from FM 1-43 FX, a fixable endocytosis marker, was quantified using confocal fluorescence imaging of whole mount sections of the organ of Corti from cochleae resected and fixed at several time points after intracochlear delivery. Intracochlear delivery of FM 1-43 FX near the base of the cochlea produces a base-apex gradient of fluorescence in the row of inner hair cells after 1 h post-delivery that is consistent with diffusion-limited transport along the scala tympani. By 3 h post-delivery there is approximately an order of magnitude decrease in peak average fluorescence intensity, suggesting FM 1-43 FX clearance from both the perilymph and inner hair cells. The increase in fluorescence intensity at 72 h post-delivery compared to 3 h post-delivery may implicate a potential radial transport pathway into the scala media., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The Mongolian gerbil is a classic animal model for age-related hearing loss. As a prerequisite for studying age-related changes, we characterized cochlear afferent synaptic morphology in young adult gerbils, using immunolabeling and quantitative analysis of confocal microscopic images. Cochlear wholemounts were triple-labeled with a hair-cell marker, a marker of presynaptic ribbons, and a marker of postsynaptic AMPA-type glutamate receptors. Seven cochlear positions covering an equivalent frequency range from 0.5 - 32 kHz were evaluated. The spatial positions of synapses were determined in a coordinate system with reference to their individual inner hair cell. Synapse numbers confirmed previous reports for gerbils (on average, 20-22 afferents per inner hair cell). The volumes of presynaptic ribbons and postsynaptic glutamate receptor patches were positively correlated: larger ribbons associated with larger receptor patches and smaller ribbons with smaller patches. Furthermore, the volumes of both presynaptic ribbons and postsynaptic receptor patches co-varied along the modiolar-pillar and the longitudinal axes of their hair cell. The gradients in ribbon volume are consistent with previous findings in cat, guinea pig, mouse and rat and further support a role in differentiating the physiological properties of type I afferents. However, the positive correlation between the volumes of pre- and postsynaptic elements in the gerbil is different to the opposing gradients found in the mouse, suggesting species-specific differences in the postsynaptic AMPA receptors that are unrelated to the fundamental classes of type I afferents., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The basolateral membrane of the mammalian inner hair cell (IHC) expresses large voltage and Ca 2+ gated outward K + currents. To quantify how the voltage-dependent activation of the K + channels affects the functionality of the auditory nerve innervating the IHC, this study adopts a model of mechanical-to-neural transduction in which the basolateral K + conductances of the IHC can be made voltage-dependent or not. The model shows that the voltage-dependent activation of the K + channels (i) enhances the phase-locking properties of the auditory fiber (AF) responses; (ii) enables the auditory nerve to encode a large dynamic range of sound levels; (iii) enables the AF responses to synchronize precisely with the envelope of amplitude modulated stimuli; and (iv), is responsible for the steep offset responses of the AFs. These results suggest that the basolateral K + channels play a major role in determining the well-known response properties of the AFs and challenge the classical view that describes the IHC membrane as an electrical low-pass filter. In contrast to previous models of the IHC-AF complex, this study ascribes many of the AF response properties to fairly basic mechanisms in the IHC membrane rather than to complex mechanisms in the synapse., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Noise exposures that result in fully reversible changes in cochlear neural threshold can cause a reduced neural output at supra-threshold sound intensity. This so-called "hidden hearing loss" has been shown to be associated with selective degeneration of high threshold afferent nerve fiber-inner hair cell (IHC) synapses. However, the electrophysiological function of the IHCs themselves in hidden hearing loss has not been directly investigated. We have made round window (RW) measurements of cochlear action potentials (CAP) and summating potentials (SP) after two levels of a 10 kHz acoustic trauma. The more intense acoustic trauma lead to notch-like permanent threshold changes and both CAP and SP showed reductions in supra-threshold amplitudes at frequencies with altered thresholds as well as from fully recovered regions. However, the interpretation of the results in normal threshold regions was complicated by the likelihood of reduced contributions from adjacent regions with elevated thresholds. The milder trauma showed full recovery of all neural thresholds, but there was a persistent depression of the amplitudes of both CAP and SP in response to supra-threshold sounds. The effect on SP amplitude in particular shows that occult damage to hair cell transduction mechanisms can contribute to hidden hearing loss. Such damage could potentially affect the supra-threshold output properties of surviving primary afferent neurons., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Noise-induced hearing loss (NIHL) affects a large number of military personnel and civilians. Regenerating inner-ear cochlear hair cells (HCs) is a promising strategy to restore hearing after NIHL. In this review, we first summarize recent transcriptome profile analysis of zebrafish lateral lines and chick utricles where spontaneous HC regeneration occurs after HC damage. We then discuss recent studies in other mammalian regenerative systems such as pancreas, heart and central nervous system. Both spontaneous and forced HC regeneration occurs in mammalian cochleae in vivo involving proliferation and direct lineage conversion. However, both processes are inefficient and incomplete, and decline with age. For direct lineage conversion in vivo in cochleae and in other systems, further improvement requires multiple factors, including transcription, epigenetic and trophic factors, with appropriate stoichiometry in appropriate architectural niche. Increasing evidence from other systems indicates that the molecular paths of direct lineage conversion may be different from those of normal developmental lineages. We therefore hypothesize that HC regeneration does not have to follow HC development and that epigenetic memory of supporting cells influences the HC regeneration, which may be a key to successful cochlear HC regeneration. Finally, we discuss recent efforts in viral gene therapy and drug discovery for HC regeneration. We hope that combination therapy targeting multiple factors and epigenetic signaling pathways will provide promising avenues for HC regeneration in humans with NIHL and other types of hearing loss., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Common causes of hearing loss in humans - exposure to loud noise or ototoxic drugs and aging - often damage sensory hair cells, reflected as elevated thresholds on the clinical audiogram. Recent studies in animal models suggest, however, that well before this overt hearing loss can be seen, a more insidious, but likely more common, process is taking place that permanently interrupts synaptic communication between sensory inner hair cells and subsets of cochlear nerve fibers. The silencing of affected neurons alters auditory information processing, whether accompanied by threshold elevations or not, and is a likely contributor to a variety of perceptual abnormalities, including speech-in-noise difficulties, tinnitus and hyperacusis. Work described here will review structural and functional manifestations of this cochlear synaptopathy and will consider possible mechanisms underlying its appearance and progression in ears with and without traditional 'hearing loss' arising from several common causes in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Inner hair cell (IHC) ribbon synapses of cochlea play important role in transmitting sound signal into auditory nerve and are sensitive to ototoxicity. However, ototoxic damage of ribbon synapses is not understood clearly. Roles of fibroblast growth factor 22 (FGF22) on synapse formation were explored under gentamycin ototoxicity. 6-week-old mice were injected intraperitoneally once daily with 50-150 mg/kg gentamicin for 10 days. Immunostaining with anti- GluR2&3/CtBP2 was used to estimate the number of ribbon synapses in the cochlea. Expression of FGF22 and myocyte enhancer factor 2D (MEF2D) was assayed with RT-PCR. Expression and localization of FGF22 protein were visualized with anti-FGF22 immunostaining. Hearing thresholds were assessed using auditory brainstem responses. Gentamicin administration caused reduction in ribbon synapse number and hearing impairment without effect on hair cells in CBA/J mouse model. Immunohistochemistry showed that FGF22 protein was expressed in IHCs, but not OHCs of cochlea. Gentamycin attenuated expression of FGF22 but enhanced expression of MEF2D. Cochlear infusion of recombinant FGF22 inhibited expression of MEF2D, preserved ribbon synapses, and restored hearing function impaired by gentamycin. FGF22 restores hearing loss through maintaining ribbon synapse number, likely via inhibition of MEF2D. Activating FGF22 might provide the conceptual basis for the therapeutic strategies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Hearing impairment is the most common human sensory deficit. Considering the sophisticated anatomy and physiology of the auditory system, disease-related failures frequently occur. To meet the demands of the neuronal circuits responsible for processing auditory information, the synapses of the lower auditory pathway are anatomically and functionally specialized to process acoustic information indefatigably with utmost temporal precision. Despite sharing some functional properties, the afferent synapses of the cochlea and of auditory brainstem differ greatly in their morphology and employ distinct molecular mechanisms for regulating synaptic vesicle release. Calyceal synapses of the endbulb of Held and the calyx of Held profit from a large number of release sites that project onto one principal cell. Cochlear inner hair cell ribbon synapses exhibit a unique one-to-one relation of the presynaptic active zone to the postsynaptic cell and use hair-cell-specific proteins such as otoferlin for vesicle release. The understanding of the molecular physiology of the hair cell ribbon synapse has been advanced by human genetics studies of sensorineural hearing impairment, revealing human auditory synaptopathy as a new nosological entity., (Copyright © 2015 Elsevier B.V. All rights reserved.)

No genes have been discovered for which expression is limited only to inner ear hair cells. This is hardly surprising, since the number of mammalian genes is estimated to be 20-25,000, and each gene typically performs many tasks in various locations. Many genes are expressed in inner ear sensory cells and not in other cells of the labyrinth. However, these genes are also expressed in other locations, often in other sensory or neuronal cell types. How gene transcription is directed specifically to hair cells is unclear. Key transcription factors that act during development can specify cell phenotypes, and the hair cell is no exception. The transcription factor ATOH1 is well known for its ability to transform nonsensory cells of the developing inner ear into hair cells. And yet, ATOH1 also specifies different sensory cells at other locations, neuronal phenotypes in the brain, and epithelial cells in the gut. How it specifies hair cells in the inner ear, but alternate cell types in other locations, is not known. Studies of regulatory DNA and transcription factors are revealing mechanisms that direct gene expression to hair cells, and that determine the hair cell identity. The purpose of this review is to summarize what is known about such gene regulation in this key auditory and vestibular cell type., (Published by Elsevier B.V.)

Shank proteins (1-3) are considered the master organizers of glutamatergic postsynaptic densities in the central nervous system, and the genetic deletion of either Shank1, 2, or 3 results in altered composition, form, and strength of glutamatergic postsynapses. To investigate the contribution of Shank proteins to glutamatergic afferent synapses of the inner ear and especially cochlea, we used immunofluorescence and quantitative real time PCR to determine the expression of Shank1, 2, and 3 in the cochlea. Because we found evidence for expression of Shank1 but not 2 and 3, we investigated the morphology, composition, and function of afferent postsynaptic densities from defined tonotopic regions in the cochlea of Shank1(-/-) mice. Using immunofluorescence, we identified subtle changes in the morphology and composition (but not number and localization) of cochlear afferent postsynaptic densities at the lower frequency region (8 kHz) in Shank1(-/-) mice compared to Shank1(+/+) littermates. However, we detected no differences in auditory brainstem responses at matching or higher frequencies. We also identified Shank1 in the vestibular afferent postsynaptic densities, but detected no differences in vestibular sensory evoked potentials in Shank1(-/-) mice compared to Shank1(+/+) littermates. This work suggests that Shank proteins play a different role in the development and maintenance of glutamatergic afferent synapses in the inner ear compared to the central nervous system., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Glutamate has been implicated in signal transmission between inner hair cells and afferent fibers of the organ of Corti. The inner hair cells are enriched in glutamate and the postsynaptic membranes express AMPA glutamate receptors. However, it is not known whether inner hair cells contain a mechanism for glutamate replenishment. Such a mechanism must be in place to sustain glutamate neurotransmission. Here we provide RT-PCR and immunofluorescence data indicating that system A transporter 1 (SLC38A1), which is associated with neuronal glutamine transport and synthesis of the neurotransmitters GABA and glutamate in CNS, is expressed in inner hair cells. It was previously shown that inner hair cells contain glutaminase that converts glutamine to glutamate. Thus, our finding that inner hair cells express a glutamine transporter and the key glutamine metabolizing enzyme glutaminase, provides a mechanism for glutamate replenishment and bolsters the idea that glutamate serves as a transmitter in the peripheral synapse of the auditory system., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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

This report summarizes recent neuropharmacological data at the IHC afferent/efferent synaptic complex: the type of Glu receptors and transporter involved and the modulation of this fast synaptic transmission by the lateral efferents. Neuropharmacological data were obtained by coupling the recording of cochlear potentials and single unit of the auditory nerve with intra-cochlear applications of drugs (multi-barrel pipette). We also describe the IHC afferent/efferent functioning in pathological conditions. After acoustic trauma or ischemia, acute disruption of IHC-auditory dendrite synapses are seen. However, a re-growth of the nerve fibres and a re-afferentation of the IHC were completely done 5 days after injury. During this synaptic repair, multiple presynaptic bodies were commonly found, either linked to the membrane or "floating" in ectopic positions. In the meantime, the lateral efferents directly contact the IHCs. The demonstration that NMDA receptors blockade delayed the re-growth of neurites suggests a neurotrophic role of NMDA receptors in pathological conditions.

During their development inner hair cells (IHCs), the primary sensory receptors in the mammalian cochlea, undergo a meticulously orchestrated series of changes in the expression of ion channels and in their presynaptic function. This review considers what we currently know about these changes in IHCs of mice and rats, which start hearing 10-12 days after birth. Just after terminal mitosis the IHCs are electrically quiescent and functionally isolated, expressing only small and slow outward K(+) currents in their basolateral membranes. By the first postnatal week the cells have acquired inward Ca(2+) and Na(+) currents that enable them to fire spontaneous action potentials at a time when the cochlea can not yet be stimulated by sound. These action potentials may be essential for normal development and survival of the IHCs themselves and of the afferent nerve fibres that synapse with them. At the onset of hearing the transition to a functionally mature sensory receptor comes about by the expression of a large and fast BK current, I(K,f), a KCNQ4 current, I(K,n), and by changes in the exocytotic machinery. Some implications of this complex developmental programme for the ideal of hair-cell regeneration in the mature mammalian cochlea are discussed.

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. Some enzymes using for IHCs dissociation affect these inactivation kinetics. IHCs were dissociated from guinea-pig cochlea by 1 mg/ml trypsin or 0.25 mg/ml protease VIII, and the properties of the K+ currents were compared using conventional whole-cell voltage-clamp recordings. TEA-sensitive potassium currents showed fast inactivation kinetics in both trypsin-dissociated cells and protease VIII-dissociated cells. The time constant of the inactivation phase in trypsin-treated cells was similar to that in protease VIII-treated cells. However, the rate of inactivation (compared by the ratio between the steady-state current and initial peak current) in protease VIII-treated cells was larger than that in trypsin-treated cells. In protease VIII-dissociated cells, the time constant of recovery from inactivation elucidated by paired-pulse protocol was 3.5 ms. Papain is another enzyme that is sometimes used for dissociating IHCs, so effects of papain were observed. Extracellular papain application (8 unit/ml) demonstrated a slight increase of the outward potassium currents.

It is important to develop an inner ear damage protocol for mice that avoids systemic toxicity and produces damage in a relatively rapid fashion, allowing for study of early cellular and molecular mechanisms responsible for hair cell death and those that underlie the lack of hair cell regeneration in mammals. Ideally, this damage protocol would reliably produce both partial and complete lesions of the sensory epithelium. We present a method for in vivo induction of hair cell damage in the mouse via placement of gentamicin-soaked Gelfoam in the round window niche of the inner ear, an adaptation of a method developed to study hair cell regeneration in chicks. A total of 82 subjects underwent the procedure. Variable doses of gentamicin were used (25, 50, 100 and 200 microg). Saline-soaked Gelfoam, sham-operations and the contralateral, non-operated cochlea were used as controls. Survival periods were 1, 3 and 14 days. Damage was assessed on scanning electron microscopy. We found that this method produces relatively rapid hair cell damage that varies with dose and can extend the entire length of the sensory epithelium. In addition, this protocol produces no systemic toxicity and preserves the contralateral ear as a control.

Tyrosine kinase receptors, including Trk A, Trk B and Trk C, participate in many different biological processes that are regulated by neurotrophic factors. Nerve growth factor (NGF)-triggered Trk A signaling is involved in growth, survival and differentiation of neurons in the central nervous system and in neural crest-derived cells. Trk A, Trk B and Trk C expression has been reported in the rat ventral cochlear nucleus. In the present study, we explored the immunocytochemical distribution of Trk A in the rodent inner ear. Rat and mouse cochleae were immunolabeled with a rabbit anti-Trk A polyclonal antibody (Chemicon) that has no reported cross-reactivity with Trk B and Trk C. In embryonic day 16 mice, no Trk A immunolabeling could be detected in the developing neuroepithelium. At postnatal day 6, weak Trk A labeling could be observed in both inner and outer hair cells. At postnatal day 12, enhanced punctate Trk A immunoexpression was present in hair cells. In adult mice and rats, intense Trk A labeling was observed in outer and inner hair cell bodies, in supporting cell bodies throughout the cochlea, and in spiral ganglion neurons. Trk A was not observed in stria vascularis, hair cell stereocilia, nor in the Trk B- and Trk C-rich cerebellum. This distribution pattern of Trk A suggests that its ligand, NGF, exerts significant trophic effects in the rodent inner ear.

To see if fibroblast growth factors (FGFs) might function in the central changes following auditory overstimulation we tracked immunostaining in the cochlear nucleus of adult mice with monoclonal antibodies to FGFs (FGF-1, FGF-2) and FGF receptor. After exposure nearly all outer hair cells died, while inner hair cell and fiber loss were restricted to a region midway along the cochlear spiral. FGFs staining in the cochlear nucleus appeared in hypertrophied astrocytes in the regions of nerve fiber degeneration only. For normal-sized astrocytes there was an increase in the number stained and the intensity of staining across all frequency domains, but not in neurons. The increases were modest at 3-7 days, pronounced at 14 days, modest again by 30 days, and back to control levels by 60 days. FGF receptor staining of neurons occurred equally in all mice, exposed or not. The findings suggest that astrocytes play a role in the central responses to acoustic overstimulation and cochlear damage, involving FGFs, possibly regulating the activity of intrinsic neurons or signaling axonal growth. Not limited to regions of cochlear nerve fiber and inner hair cell loss, the changes in FGFs may represent a reaction to outer hair cell damage which spreads broadly across the central pathways.

Aminoglycosides may induce irreversible hearing loss in both animals and humans. In order to study the nature and mechanisms underlying gentamicin-induced cell death in the inner ear, the cochlear neurosensory epithelia were dissected from guinea pigs and incubated with 0.5-10 mM gentamicin. Concentration-dependent loss of cell viability was detected by the inability of damaged cells to exclude propidium iodide. Outer hair cells were most sensitive towards gentamicin toxicity, followed by inner hair cells whereas Deiters and Hensen cells were not affected by the gentamicin concentrations used. The iron chelators 2,2'-dipyridyl and deferoxamine provided partial protection against gentamicin-induced hair cell death while the calcium chelator Quin-2 AM had no effect. Gentamicin (0.5-1 mM) induced condensation of chromatin typical for apoptosis. Using the fluorescent dye tetramethyl-rhodamine methyl ester and laser scanning microscopy we could visualize a loss of the mitochondrial membrane potential in damaged outer hair cells about 1 h before cell death occurred. Cyclosporin A, an inhibitor of the mitochondrial permeability pore, provided partial protection against gentamicin toxicity. This strongly suggests an involvement of the mitochondrial permeability transition in gentamicin-induced apoptosis.

Naturally occurring mutant mice provide an excellent model for the study of genetic malformations of the inner ear. Mice homozygous for the Bronx waltzer (bv/bv) mutation are severely hearing impaired or deaf and exhibit a 'waltzing' gait. Functional aspects of cochlear and vestibular efferents in the bv/bv mutant mouse are not well known. The present study was designed to evaluate several candidates of efferent neurotransmitters or neuromodulators including choline acetyltransferase (ChAT), gamma-aminobutyric acid (GABA), and calcitonin gene-related peptide (CGRP) in the inner ear of the bv/bv mutant mouse. Ultrastructural investigations at both light and electron microscopic level were performed. Ultrastructural morphologic evaluations of the cochlea and the vestibular end-organs were also undertaken. It is demonstrated that ChAT, GABA and CGRP immunoreactivities are present in the cochlea and in vestibular end-organs of bv/bv mutant mice. In the organ of Corti, immunoreactivity of ChAT, GABA and CGRP is confined to the inner spiral fibers, tunnel-crossing fibers, and the vesiculated nerve endings synapsing with outer hair cells. Interestingly, immunoreactivity was detectable even where inner hair cells appeared missing. Results also revealed malformations of the outer hair cells with synaptic contacts to efferent nerve endings consistently intact. In the neurosensory epithelia of the vestibular end-organs, the presence of ChAT, GABA, and CGRP immunoreactivity was localized at the vestibular efferents, with the exception of the macula of saccule. In one 8-month-old macula of utricle where the depletion of hair cells appeared highest, ChAT immunostaining was still discernible. Ultrastructural investigation demonstrated that vesiculated efferent nerve endings make synaptic contact with the outer hair cells in the organ of Corti and with type II hair cells in the vestibular end-organs. The present study provides further support that the efferent system in the bv/bv mutant inner ear is morphologically as well as functionally mature. These findings also demonstrate that if and when the onset of efferent degeneration in the bv/bv mutant inner ear occurs, it transpires subsequent to pathological conditions in the hair cells. The present findings give further indication that the efferent systems of the bv/bv mutant inner ear are independent of the afferent systems in many aspects including development, maturation as well as degeneration.

Calpains, a family of calcium-activated proteases that breakdown proteins, kinases, phosphatases and transcription factors, can promote cell death. Since leupeptin, a calpain inhibitor, protected against hair cell loss from acoustic overstimulation, we hypothesized that it might protect cochlear and vestibular hair cells against gentamicin (GM) ototoxicity. To test this hypothesis, mouse organotypic cultures from the cochlea, maculae of the utricle and the crista of the semicircular canal (P1-P3) were treated with different doses of GM (0.1-3 mM) alone or in the presence of leupeptin (0.1-3 mM). The percentage of outer hair cells (OHCs) and inner hair cells (IHCs) decreased with increasing doses of GM between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced GM-induced damage to IHCs and OHCs; this protective effect was dose-dependent. GM also significantly reduced hair cell density in the crista and utricle in a dose-dependent manner between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced hair cell loss in the crista and utricle for GM concentrations between 0.1 and 3 mM. These results suggest that one of the early steps in GM ototoxicity may involve calcium-activated proteases that lead to the demise of cochlear and vestibular hair cells.

To explore the possible involvement of glutamate (Glu) in modulation of inner hair cell (IHC) functions, the glutamate (Glu) induced changes in intracellular free Ca(2+) ([Ca(2+)]i) concentration in isolated IHCs and outer hair cells (OHCs) of the guinea pig cochlea were investigated with fluo-3, a fluorescent probe for intracellular Ca(2+). Their unique flask shape identified the IHCs with a distinct neck and spherical base with a large spherical nucleus. Normal cell shapes could be maintained for about 2 h. Fluorescence of fluo-3 was distributed in the whole isolated IHC with brighter staining nuclei. Static [Ca(2+)]i remained constant within the observation period in the absence of Glu. In the presence of a low concentration of Glu (3.85 microM), there was an increase of [Ca(2+)]i in IHCs, whereas no obvious [Ca(2+)]i change was found in OHCs. The increase of the fluorescence in IHCs reached peak level at 180 s and then gradually reduced at 400 s after the administration of Glu. The increases of [Ca(2+)]i were observed in nine of 10 IHCs, but one IHC did not show any change. For 10 of the observed OHCs, seven showed no [Ca(2+)]i change, and three showed minor reduction of [Ca(2+)]i. The increase of the Glu concentration resulted in a corresponding change of [Ca(2+)]i in the IHCs after three times administration of Glu. These results suggest that Glu acts on the IHCs presynaptic autoreceptor in a positive feedback manner.

During postnatal development of rat cochlear cells and the onset of hearing (10-23 days), the increasing endocochlear potential and energy requirements are largely provided by increased glucose utilization. It is well established that the ability of maturing rat tissues to use glucose is directly related to alteration of 6-phosphofructo-1-kinase (PFK) subunits. To gain insight into the alteration of PFK subunit levels in the cochlea from 6 to 60 days of age, PFK subunit types were measured in sections of paraffin-embedded temporal bone using IgG specific for each type of PFK subunit and quantified by computer image analysis. Although the L-type and C-type subunits did not exhibit statistically significant changes in the cochlear structures during maturation, the levels of M-type subunit in the stria vascularis cells, spiral ligament cell types I, II, and III, outer hair cells, inner hair cells, and support cells significantly increased. Also, the type IV and V spiral ligament fibrocytes during this period did not exhibit significant alterations of the M-type subunit. These data suggest that during neonatal development of the cochlear, the elevated levels of the M-type subunit are associated with increased glucose utilization and the onset of hearing.

Localization of calmodulin, a calcium binding protein, was identified in adult gerbil cochleas using paraffin section immunohistochemistry and immunogold electron microscopy with monoclonal antibody against bovine calmodulin. Immunoreactive calmodulin was abundant in inner hair cells (IHCs), outer hair cells (OHCs) and Boettcher cells of the cochleas. Other cell types containing calmodulin were marginal cells and basal cells of the stria vascularis, fibrocytes in the spiral ligament, spiral ganglion neurons and vascular smooth muscle cells. Immunogold labeling for calmodulin was observed in cuticular plate, stereocilia, and within cytoplasm of IHCs and OHCs. In OHCs the labeling was mostly observed in the region underlying lateral wall corresponding to subsurface cisterna. In IHCs the staining was diffuse in the cytoplasm and denser than that in OHCs. Boettcher cells showed dense staining along the microvillous projections facing to the intercellular spaces between Boettcher cells and Claudius cells and between the neighboring Boettcher cells. These distributions of calmodulin in the hair cells consist with the assumption that IHCs act as a true neurotransducer and OHCs as an active bi-directional mechanotransducer. The rich presence of calmodulin in Boettcher cells suggests that the cells may involve in mediating Ca(2+) regulation and play a distinctive active role in ion transport.

Tubulin, the principal component of microtubules, exists as two polypeptides, termed alpha and beta. Seven isotypes of beta tubulin are known to exist in mammals. The distributions of four beta tubulin isotypes, beta(I), beta(II), beta(III), and beta(IV), have been examined in the adult cochlea by indirect immunofluorescence using isotype-specific antibodies. In the organ of Corti, outer hair cells contained only beta(I) and beta(IV), while inner hair cells contained only beta(I) and beta(II). Inner and outer pillar cells contained beta(II) and beta(IV), but Deiters cells contained those isotypes plus beta(I). Fine fibers in the inner spiral bundle, tunnel crossing fibers, and outer spiral fibers, probably efferent in character, contained beta(I), beta(II), and beta(III), but not beta(IV). In the spiral ganglion, the somas and axons of neurons contained all four isotypes, and the myelination of ganglion cells also contained beta(I). Fibers of the intraganglionic spiral bundle contained beta(I), beta(II), and beta(III). No antibody labeled the dendritic processes of spiral ganglion neurons. The differences in isotype distribution in organ of Corti and neurons described here are consistent with and support the multi-tubulin hypothesis, which states that tubulin isotypes are expressed specifically in different cell types and may therefore have different functions.

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker's organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.

The expression of mRNA encoding plasma membrane calcium ATPase (PMCA) subunit isoforms (1-4) and splice variants was examined in the adult and developing rat cochlea by PCR and in situ hybridization. High levels of PMCA mRNA expression were observed in the neurons of the spiral ganglion, and in hair cells. Spiral ganglion neurons expressed PMCA 1-3 beginning in embryonic development, reaching high levels shortly after birth, and continuing into adulthood. Inner hair cells expressed PMCA 1 at moderate levels from birth to the time of onset of cochlear function on postnatal day 12, and strongly from then until adulthood. Outer hair cells expressed PMCA 2 at high levels from shortly after birth through adulthood. The data suggest that the calcium clearance requirements of inner and outer hair cells are distinct. PMCA 2 is the isoform with the highest affinity for calmodulin, and has also been associated with high levels of inositol triphosphate. Its presence in outer hair cells suggests that regulation of the enzyme by calmodulin may be particularly important for this hair cell type. It further suggests that inositol phosphate may play a unique role in the outer hair cell.

The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10-100 microM), consistent with the direct activation of ATP-gated ion channels. A K(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP-gated ion channels, was blocked by including 10 mM 1,2-his(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution. Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-clamp measurements of Na+ entry with regional increases in Na+-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.

Enkephalins are generally considered as neuropeptides in the central and peripheral nervous system of mammals bound to three large precursor molecules. Several animal studies demonstrated the distribution of met- and leu-enkephalin-like immunoreactivities in neurons and terminals of the lateral olivocochlear system. The immunostainings in the medial system are more controversial. No data about the presence of different enkephalin sequences in the vestibular efferent terminals are known. In the present study, the ultrastructural localization and distribution of immunoreactivities for six different antibodies against met- and leu-enkephalins in the human cochlear and vestibular periphery were investigated. A modified method of pre-embedding immunoelectronmicroscopy was applied. Met- and leu-enkephalin-like immunoreactivities were observed in the efferent terminals of the human outer and inner hair cell region. Using different met- and leu-enkephalin antibodies, the distribution of immunoreactivities remained similar. In the five human vestibular endorgans, enkephalin-like immunostaining was absent.

K+ effluxed from outer hair cells and their nerves is thought to flow laterally to strial marginal cells for recycling into scala media. Observations reported here provide evidence that K+ effluxed from inner hair cells and inner radial nerves travels medially through border cells, inner sulcus cells (ISCs), limbal fibrocytes and interdental cells (IDCs) for return to endolymph. Morphologic features of ISCs in the medial route resembled those of Hensen and Claudius cells in the lateral indicating an ion transport role for ISCs like that of Hensen and Claudius cells. Na,K-ATPase in plasmalemma of IDCs testified to their capacity to resorb and transport K+ through their known gap junctions. IDCs were differentiated into three subgroups. The most lateral IDCs formed short and long columns. Long columns contacted the medialmost ISC inferiorly and the undersurface of the tectorial membrane superiorly providing thereby a potential transcellular route for K+ transit from ISCs to endolymph. Short columns faced inner sulcus below and tectorial membrane above and accordingly possessed cells with opposite polarity at the bottom and top of the column. Short columns thus appeared situated to resorb electrolytes from limbal stroma for release into inner sulcus and beneath tectorial membrane at opposite ends of the column. The central IDCs were positioned for resorbing and transporting K+ effluxing from the Na,K-ATPase-rich stellate fibrocytes which spread toward the IDCs from near the inner sulcus. The most medial IDCs lined cuplike invaginations near the attachment of Reissner's membrane and lay apposed to light fibrocytes located between supralimbal fibrocytes and the medial IDCs. Content of Na,K-ATPase and position in the K+ transport route likened the limbal stellate fibrocytes to the spiral ligament type II fibrocytes and supralimbal fibrocytes to suprastrial fibrocytes in the lateral wall. From content of creatine kinase and position in the transport path, limbal light fibrocytes appeared analogous to spiral ligament type I fibrocytes. The additional finding that limbal fibrocytes showed unchanged or upregulated Na,K-ATPase immunoreactivity in aged gerbils with strial atrophy provided further evidence for an independent medial transport route and for the survival of inner hair cells in presbyacusis.

The inner hair cell (IHC) synapse is one of the stages of cochlear processing that determine the relation between sound pressure level and spike rate in auditory nerve fibres. Transmitter released in the non-stimulated condition is held responsible for the wide range of spontaneous spike rates (SR) observed in these fibres. Properties of stimulated spike activity in auditory nerve fibres, including rate threshold and operating range of a fibre, are known to systematically vary with SR. This paper presents a model analysis of the relation between IHC transmembrane potential and transmitter release rate as becoming manifest in these spontaneous and driven rate properties. A previously developed computational model is used to identify those transfer properties of its synapse section which lead to reproduction of the variation of rate thresholds, shapes of rate-intensity functions and maximal driven rate with SR known from the literature. First a simple additive release model, in which driven transmitter release depends linearly on IHC potential, is elaborated. Its results lead to the hypothesis that the true release function is non-linear and variable across synapses generating different SR. An exponential release function is then introduced, with parameters varying across SR in a physiologically dictated way. This approach leads to adequate reproduction of the variation in rate thresholds and rate-intensity functions with SR. Finally, the model is applied in an inverse way to directly estimate the release function from given rate-intensity functions. The conclusion of both forward and inverse model analyses is that transmitter release is a non-linear function of IHC potential which, by the systematic variation of its parameters across SR, effectively leads to the physiological variation in dynamic range across fibres of different SR. Possible relations of these results with ultrastructural morphology and basic physiology of IHC synapses are discussed.

The apices of the majority of cells of the organ of Corti are connected together by junctional complexes to form the reticular lamina, a barrier that prevents the mixing of endolymph and perilymph. These complexes include tight junctions, adherens junctions and desmosomes. Further information is required about the identity and distribution of the molecules involved in these connections if the function and organization of the reticular lamina are to be well understood. One major category of molecules occurring in adherens junctions and desmosomes, and involved in the maintenance of tissue integrity, is the cadherins. However, although cadherin has been identified in junctions between supporting cells in the adult mammalian organ of Corti at the light microscopic level, its ultrastructural distribution has not so far been described. A post-embedding immunogold labelling technique has therefore been used in conjunction with a monoclonal antibody to cadherin to investigate its ultrastructural distribution in the adult guinea-pig reticular lamina. Immunolabelling is observed in hair cell-supporting cell junctions and in supporting cell-supporting cell junctions. In addition, there is more labelling associated with inner hair cell-supporting cell junctions than with outer hair cell-supporting cell junctions. This may indicate that the junctions associated with the two types of hair cell have different functional properties.

We have previously reported the isolation from a guinea pig organ of Corti cDNA library of a cDNA clone that encodes a novel isoform of the anion exchanger 2 (AE2) protein (Negrini, Rivolta, Kalinec and Kachar, 1995. Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain. Biophys. Acta, 1236, 207-211). The deduced protein, named AE2alpha, has a conserved cytoplasmic domain and a short membrane domain with only two membrane spanning regions, as opposed to the fourteen present in the conventional AE2. Now, we are showing the immunolocalization and preliminary characterization of this protein using an antipeptide antibody specific for this novel AE2 isoform. In Western blots, this antibody binds to an approximately 89 kDa polypeptide that corresponds to a phosphorylated protein with serines as main phosphate acceptor residues. In immunofluorescence experiments, the antibody labels the stereocilia and the lateral wall of the outer hair cells and the stereocilia of the inner hair cells. Our results suggest that AE2alpha is a membrane-cytoskeletal linker in regions of the hair cell, where sensory transduction mechanisms take place.

Thyroid hormone receptors belong to the superfamily of nuclear receptors which are expressed in many tissues. Reduced levels of thyroid hormones in acquired or congenital hypothyroidism can lead to hearing loss which may be irreversible. In this study we investigated immunohistochemically the postnatal distribution of the triiodothyronine alpha receptor in the rat cochlea. Cell regions of high sensitivity towards thyroid hormones should have a high density of thyroid hormone receptors. Strong immunoreactivity for the alpha-thyroid hormone receptor was observed in spiral ganglion cells as well as inner and outer hair cells of the cochlea. Staining could be detected during all stages investigated from the first postnatal day up to day 30 and exhibited mainly a nuclear pattern. These observations suggest that spiral ganglion cells and hair cells are target regions of thyroid hormones in the adult and developing cochlea. Thyroid hormones could thus play an important role in the maturation of the inner ear.

Stains with antibodies to glutamic acid decarboxylase (GAD) and gamma-aminobutyric acid (GABA) in the cochlea of postnatal and adult mice reveal within the inner spiral bundle a distinctive neuronal plexus intimately associated with the inner hair cells. This innervation provides endings that cradle the receptor poles of the sensory cells and lateral end collaterals that wind between the cells, distributing endings alongside and around them. Some GAD-positive fibers enter the inner pillar bundle, from where they distribute tunnel fibers to the outer hair cells and recurrent collaterals to the inner hair cells. The GABAergic innervation within the inner spiral bundle is present along the entire cochlear axis, with the highest density in its basal half. Stainings against calcitonin gene-related peptide (CGRP), which localizes in the cholinergic counterpart of the inner spiral bundle, reveal that some of these fibers parallel the GABAergic circuit. The present data, together with our previous demonstration of compound (serial, converging, triadic) efferent synapses within this pathway (Sobkowicz et al. (1995) Abst. ARO 18, 171) evidences the presence of a distinctive innervation to the inner hair cells, hitherto unrecognized. The expression of growth-associated protein-43 (GAP-43) within the inner hair cell innervation in the adult cochlea provides evidence for a continuous synaptic turnover and plasticity, thus emphasizing its functional importance.

Post-translational modifications to tubulin in the sensory and supporting cells of the cochlea were studied using antibodies specific to the tyrosinated, detyrosinated, acetylated and polyglutamylated isoforms. In the sensory cells, microtubules which label intensely with antibodies to tyrosinated tubulin are found in networks within the cytoplasm. Microtubules which label with antibodies to detyrosinated tubulin and polyglutamylated tubulin, but not acetylated tubulin, form a small component of the microtubules found in the cytoplasm only in the region below the cuticular plate. Microtubules in the supporting cells (inner and outer pillar cells and Deiters cells) are arranged in bundles and contain little tyrosinated tubulin. They are composed instead of predominantly post-translationally modified isoforms which include detyrosinated, acetylated and polyglutamylated tubulin. The findings suggest that microtubules in the sensory cells form dynamic structures, since microtubules that undergo cyclic polymerization and depolymerization predominantly contain tubulin that has not yet had its carboxy-terminal tyrosine residue removed. The presence of microtubules in the supporting cells in which the tubulin has been polymerized into microtubules long enough to be post-translationally modified, provides evidence that these microtubules are stable, long-lived and could contribute to the structural support of the sensory organ of Corti.

The expression of fimbrin in the developing rat cochlea was analyzed using an immunohistochemical technique with fimbrin antibody. The cochlea displayed temporal and lateral-longitudinal gradients for fimbrin expression during development. Fimbrin immunoreactivity first appeared in the inner hair cell stereocilia of the basal turn on the first gestational day studied (day 18). At birth, both inner (IHC) and outer hair cell (OHC) stereocilia of the basal turn showed positive labeling with fimbrin antibody. The progression of appearance was always from IHCs to OHCs and fimbrin immunostaining appeared in the apical hair cells by postnatal day 6. Immunostaining was restricted to stereocilia and the cuticular plate, and no immunoreactivity was observed in neighboring structures of the epithelium. Double labeling using both fimbrin antibody and phalloidin binding revealed similar chronological expression from the earliest stage studied. Increasing fimbrin immunoreactivity was observed in hair cells until late postnatal and adult stages. This study suggests that fimbrin is expressed with F-actin during development and fimbrin together with actin may constitute the two basic molecules that participate in stereocilia formation. We speculate that fimbrin may help maintain the parallel growth of actin filaments within the stereocilia. These data additionally support previous findings that hair cell maturation occurs from the base to the apex and from IHCs to OHCs.

Two different radioligands were used to identify extracellular ATP binding sites specific to P2 purinoceptors in guinea-pig cochlear tissue. Deoxyadenosine 5'-(alpha-[35S]thio)triphosphate ([35S]dATP alpha S; 10 nM) provided a high activity probe for the P2y purinoceptor subtype on the basis of selective block by 2-methylthio-ATP (2MeSATP; 100 microM). [3H]alpha, beta-methylene-ATP (10 nM), a high affinity probe for a P2x purinoceptor subtype was selectively blocked by inclusion of the related compound beta, gamma-methylene-ATP (100 microM). Both probes labelled the organ of Corti, stria vascularis and spiral prominence regions. The P2x purinoceptor probe also bound to lateral wall tissue below the spiral prominence and insertion point of the basilar membrane within the scala tympani compartment, a region which failed to show significant binding using [35S]dATP alpha S. Frozen sections of whole cochlea permitted analysis of radioligand binding to the cell body region (spiral ganglion in Rosenthal's canal) of the primary auditory afferents and the auditory nerve itself, which lies within the central region of the modiolus of the cochlea. Both these regions exhibited 2MeSATP blockable [35S]dATP alpha S binding whereas specific [3H]alpha, beta-methylene-ATP binding was absent from spiral ganglion and minimal in the auditory nerve region. These results demonstrate a mixed P2 purinoceptor distribution in cochlear tissues and suggest that complex purine-mediated neurohumoral mechanisms may influence cochlear function at a number of sites.

The composition of cytoskeletal elements in hair cells and non-sensory cells was studied in paraformaldehyde fixed cochleae of the horseshoe bat and the gerbil using phallotoxins and antibodies directed against actin, alpha-tubulin and fodrin. In both species, cryostat sections of the organ of Corti were studied using confocal fluorescence microscopy; in the bat, ultrathin sections were investigated using actin-immunoelectron and classical electron microscopy. F-actin was found in stereocilia and cuticular plates of inner and outer hair cells (IHCs and OHCs) of both species. In fixed material from both species, no F-actin staining was detected in the cytoplasm or along the lateral cell membrane of OHCs, whereas in freshly isolated OHCs of the gerbil, a faint F-actin staining was detected along the lateral wall. In the bat, the patterns of F-actin staining were confirmed with actin-immunoelectron microscopy. The alpha-tubulin antibody strongly labeled IHCs of both species. They contained a complex network of microtubules especially in the neck portion. In the bat, OHCs showed no distinct alpha-tubulin reactivity, as would be expected given the scarcity of microtubules observed at the ultrastructural level. In the gerbil, alpha-tubulin reactivity was found throughout the OHC body with highest intensity in the cell apex. In Deiters cells, pillar cells and Boettcher cells of both species, F-actin and microtubules were colocalized at contact zones with the basilar membrane. In Deiters cups, F-actin staining was most pronounced in the basal turn of the bat cochlea. In the gerbil, a distinct baso-apical gradient was found in immunostaining properties and morphology of the Deiters cells. Intense fodrin reactivity was found in the cuticular plates and along the lateral cell membrane of both types of hair cells of the bat. Cytoplasmic fodrin staining was localized within the IHCs of the bat. In the gerbil, intense fodrin staining was only found in cuticular plates of hair cells and staining of the lateral cell membrane of hair cells was faint. A faint fodrin staining was also seen in Deiters cells of both species. The basic arrangement of the cytoskeletal elements in the batś organ of Corti is similar to that of other mammals, however, certain features suggest the presence of subtle differences in micromechanical properties: there is an increased concentration of microtubules in the neck portion of IHCs, an increase in the amount of F-actin within the Deiters cups and a reduced amount of microtubules in the OHCs.

The distribution of amikacin (AK), an exclusive cochleo-toxic aminoglycosidic antibiotic (AA), and of gentamicin (GM), which is both cochleo- and vestibulo-toxic, has been studied in cochlear and vestibular hair cells. Guinea pigs were treated during six days with one daily injection of AK (450 mg/kg/day) or GM (60 mg/kg/day). AAs were detected, using immunocytochemical technique with scanning laser confocal microscopy, in isolated cells from guinea pigs sacrificed from 2 to 30 days after the end of the treatments. Results demonstrate a rapid uptake (as soon as after 2-day treatment) of both AAs by cochlear and vestibular hair cells and a very slow clearance. Particularly GM and AK are detected in type I and type II hair cells of the utricles and cristae ampullaris. The presence of these two molecules with different toxic potentialities towards cochlear and vestibular hair cells indicates that the selective ototoxicity of aminoglycosides cannot be explained simply on the basis of particular uptake and accumulation in the different sensory hair cells.

The distribution of the two cytoskeletal proteins, filamentous actin (F-actin) and fodrin, was investigated along the organ of Corti of the chinchilla using laser scanning confocal fluorescence microscopy. High intensity labeling of F-actin was seen in outer and inner hair cells, including the stereocilia. High intensity staining was also seen for fodrin in outer and inner hair cells, but not in their stereocilia. Staining intensity of both proteins along the lateral cell wall of the outer hair cells appeared to be greater in the middle and basal cochlear turns than in the apical turn. Pillars and Deiters cells also exhibited high intensity labeling of F-actin. The lack of significant differences in the distribution of fodrin between outer and inner hair cells makes the role of this protein in the active processes still unclear. Comparison of the distribution of F-actin and fodrin in the chinchilla with those reported in the guinea pigs suggest possible species differences.

The localization of subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) selective glutamate receptor, termed Glutamate receptor (GluR) was examined in the rat cochlea using affinity purified polyclonal antibody to GluR subunits (GluR 1-4). GluR 2/3 and GluR 4 immunoreactive (IR) staining was observed in rat spiral ganglion cells, while GluR 1 IR was not. GluR 4 IR staining was also seen in puncta at inner and outer hair cell bases. These results suggest that GluR 2/3 and GluR 4 are components of excitatory amino acid synapses in the rat cochlea.