This composite article is intended to give the experts in the field of cochlear mechanics an opportunity to voice their personal opinion on the one mechanism they believe dominates cochlear amplification in mammals. A collection of these ideas are presented here for the auditory community and others interested in the cochlear amplifier. Each expert has given their own personal view on the topic and at the end of their commentary they have suggested several experiments that would be required for the decisive mechanism underlying the cochlear amplifier. These experiments are presently lacking but if successfully performed would have an enormous impact on our understanding of the cochlear amplifier.

This chapter reviews the molecular composition and structure of hair cells and supporting cells in both auditory and vestibular epithelia with an emphasis on the mammalian cochlea. The anatomy of the cochlea including diagrams and micrographs of the cytoarchitecture of the sensory epithelium, the organ of Corti, is described. Details of the ultrastructure of the sensory hair cells are provided in relation to current theories of mechanotransduction, adaptation, and cochlear amplification; the main categories of ion channels and cytoskeletal, membrane, and calcium-handling proteins are discussed in relation to each of these processes. The mechanical contribution of the supporting cells associated with hair cells is also discussed along with their potential for uptake of the neurotransmitter, glutamate, which is released onto the cochlear afferents by the hair cells. The presence of ion channels, gap junctions, and water channels in the supporting cells is also mentioned briefly in relation to homeostatic support for the hair cells, in particular, the recycling of the potassium ions that carry the majority of the mechanotransduction current through the hair cells.

The tip links between stereocilia of acousticolateral hair cells have been suggested to contain cadherin 23 (CDH23) comprising an upper branched portion that is bound to a lower portion composed of protocadherin 15 (PCDH15). The molecular conformation of CDH23, its binding to PCDH15, the tip links, and mechanoelectrical transduction have all been shown previously to be sensitive to exposure to low levels of calcium. The aim of this study was to compare the characteristics of tip links in guinea-pig cochlear hair cells with reported features of the CDH23-PCDH15 complex. Tip links were examined using field emission scanning electron microscopy and transmission electron microscopy in conventional preparations and after treatment with the detergent Triton-X-100 or varying calcium concentrations in the extracellular solution. The results showed that tip links have a twisted double-stranded appearance with a branched upper region. They survived demembranation of the stereocilia by detergent suggesting that they have transmembrane domains at both ends. Their lengths, when fixed in the presence of 2 mM extracellular calcium, were approximately 150 nm. With prior exposure to 1 mM calcium their lengths were approximately 164 nm. The lengths in 50 microM calcium are similar ( approximately 185 nm) to those reported for CDH23-PCDH15 complexes in 100 microM calcium ( approximately 180 nm). Exposure to 1 microM calcium caused loss of tip links and an increased distance between the residual attachment sites. The data indicate that extracellular calcium concentration affects tip-link length. One model compatible with the recently proposed tip-link structure is that the CDH23 double strand undergoes calcium-dependent unfolding, changing the length of the links. The bundle may also tilt in the direction of the tallest row of stereocilia as the tip link lengthens and then is lost. Overall, our data are consistent with a tip link composed of complexes of CDH23 and PCDH15 but do not rule out other possibilities.

Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing β- and γ-actin isoforms which have been described as having differing intracellular distributions in chick cochlear hair cells. Here, we have used post-embedding immunogold labelling for β- and γ-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of β-actin decrease and γ-actin increase in the order, outer pillar cells, inner pillar cells, Deiters’ cells and hair cells. There is also more β-actin and less γ-actin in outer pillar cells in higher than lower frequency regions. In hair cells, β-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially in the rootlets and towards the periphery of their shafts; labelling densities for γ-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest β-actin and γ-actin form homomeric filaments. These data confirm differential distribution of these actin isoforms in the mammalian cochlea and reveal systematic differences between sensory and supporting cells. Increased expression of β-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.

Spherical bushy neurons in the anteroventral cochlear nucleus receive glutamatergic primary terminals from the cochlear nerve and terminals of noncochlear (i.e. nonprimary) origin, many of which colocalize gamma-aminobutyric acid (GABA) and glycine. Here the relationship between GABA and glycine in these terminals has been investigated using postembedding immunogold labelling. A significant negative correlation was found between the density of terminal labelling for GABA and for glycine in four guinea pigs. Terminals could be divided into three categories, GABA-only, glycine-only, or colocalizing depending on whether they had a significantly higher labelling density for either amino acid than the primary terminals, The overall labelling density in all four animals was significantly greater for GABA in GABA-only terminals than colocalizing ones but similar for glycine in both. Within the terminals, the labelling density over synaptic vesicles, nonvesicular regions of cytoplasm and mitochondria was also investigated. No significant difference was detected in the labelling density of vesicles compared with nonvesicular regions for either amino acid. However, a significant difference was found between the overall labelling density over mitochondria and nonvesicular regions for both. There was also significantly more mitochondrial GABA labelling in GABA-only terminals compared to colocalizing terminals but mitochondrial glycine labelling was similar in glycine-only and colocalizing terminals. Thus the level of GABA is higher in single than in colocalizing terminals, particularly in the mitochondria, but similar for glycine in both. It is possible therefore that the presence of glycine affects the level of GABA in the nonprimary terminals but that the presence of GABA does not affect the level of glycine.

Double labeling is used for localizing two antigens simultaneously in the same tissue. We have used two approaches to post-embedding immunogold labeling to investigate whether nerve terminals in the guinea-pig anteroventral cochlear nucleus (AVCN) that contain γ-aminobutyric acid (GABA) or glycine are capable of retrieving the other amino acid as part of an investigation of colocalization of these putative neurotransmitters. For this, vibroslices of perfusion-fixed brain stem were freeze-substituted and embedded in the low temperature resin, Lowicryl HM20. Simultaneous labeling of ultrathin sections was then performed with a mixture of a rabbit primary antibody to GABA and a guinea-pig primary antibody to the glycine transporter, GLYT2, followed by labeling with a mixture of secondary antibodies (goat anti-rabbit IgG-30 nm gold, goat anti-guinea pig IgG-15 nm gold). This approach indicated that GLYT2 occurs in the plasma membrane of some terminals that contain GABA. The other approach involved sequential labeling of ultrathin sections with a rabbit primary antibody to the GABA transporter, GAT1, followed by an anti-rabbit secondary antibody conjugated to 15-nm gold particles. Sections were then treated with paraformaldehyde vapor to denature any free anti-IgG binding sites on the first antibody, and labeled with a primary antibody to glycine also raised in rabbit followed by an anti-rabbit secondary antibody conjugated to 30-nm gold particles. This approach indicated that GAT1 occurs in the plasma membrane of some terminals that contain glycine. Thus, these techniques can be used to localize heat-labile multiple antigens in the same tissue.

Calmodulin is found in the mechanosensitive stereociliary bundle of hair cells where it plays a role in various calcium-sensitive events associated with mechanoelectrical transduction. In this study, we have investigated the ultrastructural distribution of calmodulin in the apex of guinea-pig cochlear hair cells, using post-embedding immunogold labelling, in order to determine in more detail where calmodulin-dependent processes may be occurring. Labelling was found in the cuticular plate as well as the hair bundle, the rootlets of the stereocilia being more densely labelled than the surrounding filamentous matrix. In the bundle, labelling was found almost exclusively at the periphery rather than over the centre of the actin core of the stereocilia, and was clearly associated with the attachments of the lateral links that connect them to their nearest neighbours. It was also found to be denser towards the tips of stereocilia compared to other stereociliary regions and occurred consistently at either end of the tip link that connects stereocilia of adjacent rows. The contact region between stereocilia that is found just below the tip link was also clearly labelled. These concentrations of labelling in the bundle are likely to indicate sites where calmodulin is associated with calcium/calmodulin-sensitive proteins such as the various myosin isoforms and the plasma membrane ATPase (PMCA2a) that are known to occur there, and possibly with the transduction channels themselves. At least one of the myosin isoforms, myosin 1c, is thought to be associated with slow adaptation, and PMCA2a with control of calcium levels in the bundle. The concentration of calmodulin in the contact region further supports the suggestion that this is a functionally distinct region rather than a simple geometrical association between adjacent stereocilia.

OBJECTIVES:The aims of this study are (1) to investigate if significant long-term recovery of mature hair bundle (MHB) numbers takes place following gentamicin-induced damage to the mammalian vestibular sensory epithelium and (2) to assess if the different MHB types in the vestibular sensory epithelium have a different susceptibility to ototoxic damage.METHODS:Gentamicin (8 mg in 0.1-mL sterile water) was injected transtympanically into one ear of guinea pigs, the contralateral ear acting as a control. The animals were killed at 4 days, 4 weeks, and 3 and 10 months post-treatment and the utricles (n = 38) were extracted from both ears. Mature hair bundle and immature-looking hair bundle (IHB) densities on the surface of the utricle were determined using scanning electron microscopy.RESULTS:The MHB density showed a significant decline between 4 days and 4 weeks post-treatment. There was greater loss of type I MHBs (tallest stereocilia comparable in height to the kinocilium) than type II MHBs (kinocilium taller than the tallest stereocilia). A significant increase in IHB density was seen at 4 weeks post-treatment, after which it declined rapidly. A significant but incomplete recovery in MHB density (to 66% of control value) was seen in the striolar region at 10 months post-treatment, and these were composed mainly of type II MHBs.CONCLUSIONS:It would appear that the mature mammalian vestibular sensory epithelium does have the capacity for long-term recovery of MHB numbers following gentamicin-induced damage, but this is limited and does not result in complete restoration of the epithelium. Type I MHBs are more susceptible to ototoxic damage than type II MHBs. Sommaire

Clinical Otolaryngology & Allied SciencesVolume 25, Issue 1 p. 86-86 Is Electronystagmography of any Diagnostic Value in the Elderly? D. Hajiof, D. Hajiof Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorR.M. Barr-Hamilton, R.M. Barr-Hamilton Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorN.R. Colledge, N.R. Colledge Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorS.J. Lewis, S.J. Lewis Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorJ.A. Wilson, J.A. Wilson Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this author D. Hajiof, D. Hajiof Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorR.M. Barr-Hamilton, R.M. Barr-Hamilton Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorN.R. Colledge, N.R. Colledge Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorS.J. Lewis, S.J. Lewis Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this authorJ.A. Wilson, J.A. Wilson Department of Otolaryngology, Freeman Hospital, Newcastle-upon-Tyne,Search for more papers by this author First published: 10 August 2009 https://doi.org/10.1046/j.1365-2273.2000.00337-4_25_1.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume25, Issue1February 2000Pages 86-86 RelatedInformation

Clinical Otolaryngology & Allied SciencesVolume 24, Issue 1 p. 82-89 Otorhinolaryngology Research Society Abstracts Mammalian vestibular hair-cell regeneration R. M. Walsh, R. M. Walsh (University Hospital Birmingham andSearch for more papers by this authorC. M. Hackney, C. M. Hackney Department of Communication and Neuroscience, Keele University, UK)Search for more papers by this authorD. N. Furness, D. N. Furness Department of Communication and Neuroscience, Keele University, UK)Search for more papers by this author R. M. Walsh, R. M. Walsh (University Hospital Birmingham andSearch for more papers by this authorC. M. Hackney, C. M. Hackney Department of Communication and Neuroscience, Keele University, UK)Search for more papers by this authorD. N. Furness, D. N. Furness Department of Communication and Neuroscience, Keele University, UK)Search for more papers by this author First published: 25 December 2001 https://doi.org/10.1046/j.1365-2273.1999.00196-15.xRead the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume24, Issue1February 1999Pages 82-89 RelatedInformation

Clinical Otolaryngology & Allied SciencesVolume 24, Issue 1 p. 82-89 Otorhinolaryngology Research Society Abstracts Morphological effects of low extracellular calcium on stereociliary bundles in the guinea-pig cochleacm B. N. Kumar, B. N. KumarSearch for more papers by this authorD. N. Furness, D. N. FurnessSearch for more papers by this authorC. M. Hackney, C. M. HackneySearch for more papers by this author B. N. Kumar, B. N. KumarSearch for more papers by this authorD. N. Furness, D. N. FurnessSearch for more papers by this authorC. M. Hackney, C. M. HackneySearch for more papers by this author First published: 25 December 2001 https://doi.org/10.1046/j.1365-2273.1999.00196-6.xRead the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume24, Issue1February 1999Pages 82-89 RelatedInformation

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.

The mechanosensitive transducer channels of hair cells have long been proposed to be gated directly by tension in the tip links. These are thin, elastic extracellular elements connecting the tips of adjacent stereocilia located on the apical surface of the cell. If this hypothesis is true, the channels should close after destruction of tip links. The hypothesis was tested pharmacologically using receptor currents obtained in response to mechanical stimulation of the stereociliary bundle of outer hair cells isolated from the adult guinea pig cochlea. Application of elastase (20 U/ml) or 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA; 5 mm), both of which are known to disrupt tip links in other hair-cell preparations, led to the expected irreversible loss of receptor currents. However, the cells then displayed a maintained inward current, implying that channels were left permanently open. This current was similar in magnitude to the receptor current before treatment and was reduced reversibly by known blockers of mechanosensitive channels, namely, dihydrostreptomycin (100 μm), amiloride (300 μm), and gadolinium ions (1 mm). These observations suggest that the maintained current flows through the mechanosensitive channels. Electron microscopical analysis of isolated hair cells, exposed to the same concentrations of elastase or BAPTA as in the electrophysiological experiments, demonstrated an almost total loss of tip links in hair bundles that showed no evidence of other mechanical damage. It is concluded that although the tip links are required for mechanoelectrical transduction, the channels are not gated directly by the tip links.