Electrical and Immunohistochemical Properties of Cochlear Fibrocytes in 3D Cell Culture and in the Excised Spiral Ligament of Mice.

Fibrocyte degeneration in the cochlear lateral wall is one possible pathology of age-related metabolic hearing loss (presbycusis). Within the lateral wall fibrocytes play a role in potassium recycling and maintenance of the endocochlear potential. It has been proposed that cell replacement therapy could prevent fibrocyte degeneration in the CD/1 mouse model of hearing loss. For this to work, the replacement fibrocytes would need to take over the structural and physiological role of those lost. We have grown lateral wall fibrocytes from neonatal CD/1 mice in a 3D-collagen gel culture with the aim of assessing their functional similarity to native lateral wall fibrocytes, the latter in a slice preparation and in excised spiral ligament pieces. We have compared cultured and native fibrocytes using both immuno-labelling of characteristic proteins and single cell electrophysiology. Cultured fibrocytes exhibited rounded cell bodies with extending processes. They labelled with marker antibodies targeting aquaporin 1 and calcium-binding protein S-100, precluding an unambiguous identification of fibrocyte type. In whole-cell voltage clamp, both native and cultured fibrocytes exhibited non-specific currents and voltage-dependent K+ currents. The non-specific currents from gel-cultured and excised spiral ligament fibrocytes were partially and reversibly blocked by external TEA (10 mM). The TEA-sensitive current had a mean reversal potential of + 26 mV, suggesting a permeability sequence of Na+ > K+. These findings indicate that 3D-cultured fibrocytes share a number of characteristics with native spiral ligament fibrocytes and thus might represent a suitable population for transplantation therapy aimed at treating age-related hearing loss.

Neuronal vs glial glutamate uptake: Resolving the conundrum.

Neither normal brain function nor the pathological processes involved in neurological diseases can be adequately understood without knowledge of the release, uptake and metabolism of glutamate. The reason for this is that glutamate (a) is the most abundant amino acid in the brain, (b) is at the cross-roads between several metabolic pathways, and (c) serves as the major excitatory neurotransmitter. In fact most brain cells express glutamate receptors and are thereby influenced by extracellular glutamate. In agreement, brain cells have powerful uptake systems that constantly remove glutamate from the extracellular fluid and thereby limit receptor activation. It has been clear since the 1970s that both astrocytes and neurons express glutamate transporters. However the relative contribution of neuronal and glial transporters to the total glutamate uptake activity, however, as well as their functional importance, has been hotly debated ever since. The present short review provides (a) an overview of what we know about neuronal glutamate uptake as well as an historical description of how we got there, and (b) a hypothesis reconciling apparently contradicting observations thereby possibly resolving the paradox.

Quantitative analysis of the expression of the glutamate-aspartate transporter and identification of functional glutamate uptake reveal a role for cochlear fibrocytes in glutamate homeostasis.

There are several subtypes of fibrocyte in the spiral ligament and spiral limbus of the cochlea that may contribute to fluid homeostasis. Immunocytochemical data suggest that these fibrocytes possess the glutamate-aspartate transporter, GLAST, as do supporting cells around the hair cells. However, functional glutamate uptake has not been demonstrated in fibrocytes. We used confocal and post-embedding immunogold electron microscopy to confirm that GLAST is expressed in adult fibrocytes of CD-1 mice with a relative expression: spiral limbus fibrocytes>type II>V>IV>I spiral ligament fibrocytes. Because they were sparsely present in most samples, type III fibrocytes were assessed only in one sample where their GLAST levels were similar to type I. Type II, type V and spiral limbus fibrocytes have many fine cellular processes that increase their surface area, those of the latter two coming into direct contact with perilymph, and type V fibrocytes contain the most glutamate. These data imply that glutamate uptake occurs in the fibrocytes. We assessed uptake of D-aspartate (a glutamate analogue) together with GLAST expression immunocytochemically and electrophysiologically. D-aspartate accumulated into GLAST expressing fibrocytes in vitro and evoked currents blockable by the GLAST inhibitor D,L-threo-beta-benzyloxyaspartate (TBOA), similar to those of supporting cells around inner hair cells. Currents were strongest in spiral limbus fibrocytes, progressively lower in type V and type II fibrocytes, and were negligible in type I fibrocytes in accordance with the relative expression levels of GLAST. We conclude that in addition to their known homeostatic functions, fibrocytes, in particular spiral limbus, type II and type V fibrocytes play a role in glutamate homeostasis in the cochlea.

A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: new insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2).

The relative distribution of the excitatory amino acid transporter 2 (EAAT2) between synaptic terminals and astroglia, and the importance of EAAT2 for the uptake into terminals is still unresolved. Here we have used antibodies to glutaraldehyde-fixed d-aspartate to identify electron microscopically the sites of d-aspartate accumulation in hippocampal slices. About 3/4 of all terminals in the stratum radiatum CA1 accumulated d-aspartate-immunoreactivity by an active dihydrokainate-sensitive mechanism which was absent in EAAT2 glutamate transporter knockout mice. These terminals were responsible for more than half of all d-aspartate uptake of external substrate in the slices. This is unexpected as EAAT2-immunoreactivity observed in intact brain tissue is mainly associated with astroglia. However, when examining synaptosomes and slice preparations where the extracellular space is larger than in perfusion fixed tissue, it was confirmed that most EAAT2 is in astroglia (about 80%). Neither d-aspartate uptake nor EAAT2 protein was detected in dendritic spines. About 6% of the EAAT2-immunoreactivity was detected in the plasma membrane of synaptic terminals (both within and outside of the synaptic cleft). Most of the remaining immunoreactivity (8%) was found in axons where it was distributed in a plasma membrane surface area several times larger than that of astroglia. This explains why the densities of neuronal EAAT2 are low despite high levels of mRNA in CA3 pyramidal cell bodies, but not why EAAT2 in terminals account for more than half of the uptake of exogenous substrate by hippocampal slice preparations. This and the relative amount of terminal versus glial uptake in the intact brain remain to be discovered.

The dimensions and structural attachments of tip links in mammalian cochlear hair cells and the effects of exposure to different levels of extracellular calcium.

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.

Specificity of antibodies: unexpected cross-reactivity of antibodies directed against the excitatory amino acid transporter 3 (EAAT3).

UNLABELLED: Specific antibodies are essential tools for identifying individual proteins in biological samples. While generation of antibodies is often straightforward, determination of the antibody specificity is not. Here we illustrate this by describing the production and characterization of antibodies to excitatory amino acid transporter 3 (EAAT3). We synthesized 13 peptides corresponding to parts of the EAAT3 sequence and immunized 6 sheep and 30 rabbits. All sera were affinity purified against the relevant immobilized peptide. Antibodies to the peptides were obtained in almost all cases. Immunoblotting with tissue extracts from wild type and EAAT3 knockout animals revealed that most of the antibodies did not recognize the native EAAT3 protein, and that some recognized other proteins. Several immunization protocols were tried, but strong reactions with EAAT3 were only seen with antibodies to the C-terminal peptides. In contrast, good antibodies were obtained to several parts of EAAT2. EAAT3 was only detected in neurons. However, rabbits immunized with an EAAT3-peptide corresponding to residues 479-498 produced antibodies that labeled axoplasm and microtubules therein particularly strongly. On blots, these antibodies recognized both EAAT3 and a slightly smaller, but far more abundant protein that turned out to be tubulin. The antibodies were fractionated on columns with immobilized tubulin. One fraction contained antibodies apparently specific for EAAT3 while another fraction contained antibodies recognizing both EAAT3 and tubulin despite the lack of primary sequence identity between the two proteins. Addition of free peptide to the incubation solution blocked immunostaining of both EAAT3 and tubulin. CONCLUSIONS: Not all antibodies to synthetic peptides recognize the native protein. The peptide sequence is more important than immunization protocol. The specificity of an antibody is hard to predict because cross-reactivity can be specific and to unrelated molecules. The antigen preabsorption test is of little value in testing the specificity of affinity purified antibodies.

Auditory hair cell-afferent fiber synapses are specialized to operate at their best frequencies.

Auditory afferent fiber activity is driven by high-fidelity information transfer from the sensory hair cell. Presynaptic specializations, posited to maintain fidelity, are investigated at synapses with characteristic frequencies of 120 Hz and 320 Hz. Morphological data indicate that high-frequency cells have more synapses and higher vesicle density near dense bodies (DBs). Tracking vesicular release via capacitance changes identified three overlapping kinetic components of release corresponding to morphologically identified vesicle pools. High-frequency cells released faster; however, when normalized to release site number, low-frequency cells released faster, likely due to a greater Ca2+ load per synapse. The Ca(2+)-dependence of release was nonsaturating and independent of frequency, suggesting that release, not refilling, was rate limiting. A model of release derived from vesicle equilibration between morphologically defined pools reproduced the capacitance data, supporting a critical role in vesicle trafficking for DBs. The model suggests that presynaptic specializations enable synapses to operate most efficiently at their characteristic frequencies.

Differential distribution of beta- and gamma-actin in guinea-pig cochlear sensory and supporting cells.

Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing beta- and gamma-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 beta- and gamma-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of beta-actin decrease and gamma-actin increase in the order, outer pillar cells, inner pillar cells, Deiters' cells and hair cells. There is also more beta-actin and less gamma-actin in outer pillar cells in higher than lower frequency regions. In hair cells, beta-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 gamma-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest beta-actin and gamma-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 beta-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.

An immunogold investigation of the distribution of calmodulin in the apex of cochlear hair cells.

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.

Localization of the glutamate-aspartate transporter, GLAST, in rat taste buds.

A number of putative neurotransmitter substances have been found in vertebrate taste buds. Amongst these glutamate has been localized in fibres innervating the buds and uptake of glutamate has been shown to occur into receptor cells. It is therefore possible that, in common with other sensory systems, glutamate is a neurotransmitter in taste buds. In the inner ear and retina of mammals, the membranes of supporting cells have been shown to contain the glial glutamate transporter GLAST. In the brain, this protein is involved in glutamate re-uptake into glial cells where the glutamate is converted into glutamine for recycling into glutamatergic terminals. In this study, the presence of GLAST has been investigated in taste buds in the rat vallate papilla and its distribution compared with that of glutamine to determine whether there are cells in this system that play a glia-like role in glutamate handling. Immunofluorescent labelling showed that a subset of cells in the taste bud contains GLAST. Immunogold labelling indicated that it occurs in the plasma membranes of supporting cells, especially on the fine cytoplasmic processes of dark cells towards the basal region of the bud. A protein of molecular mass similar to that of cerebellar GLAST was detected in immunoblots of excised papillae. Double labelling and semiquantitative analysis of glutamine and GLAST immunoreactivity showed that the GLAST-positive cells have a higher level of cytoplasmic glutamine than the adjacent cells. It is proposed that these GLAST-positive cells play a glia-like role in the uptake of glutamate following its release at synapses within the taste bud although the precise location of the latter remains uncertain. The GLAST-positive cells may also be involved in its subsequent conversion to glutamine in a glutamate/glutamine cycle similar to that described in the brain.

Regeneration of the mammalian vestibular sensory epithelium following gentamicin-induced damage.

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

Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti.

Hearing loss is most often the result of hair-cell degeneration due to genetic abnormalities or ototoxic and traumatic insults. In the postembryonic and adult mammalian auditory sensory epithelium, the organ of Corti, no hair-cell regeneration has ever been observed. However, nonmammalian hair-cell epithelia are capable of regenerating sensory hair cells as a consequence of nonsensory supporting-cell proliferation. The supporting cells of the organ of Corti are highly specialized, terminally differentiated cell types that apparently are incapable of proliferation. At the molecular level terminally differentiated cells have been shown to express high levels of cell-cycle inhibitors, in particular, cyclin-dependent kinase inhibitors [Parker, S. B., et al. (1995) Science 267, 1024-1027], which are thought to be responsible for preventing these cells from reentering the cell cycle. Here we report that the cyclin-dependent kinase inhibitor p27(Kip1) is selectively expressed in the supporting-cell population of the organ of Corti. Effects of p27(Kip1)-gene disruption include ongoing cell proliferation in postnatal and adult mouse organ of Corti at time points well after mitosis normally has ceased during embryonic development. This suggests that release from p27(Kip1)-induced cell-cycle arrest is sufficient to allow supporting-cell proliferation to occur. This finding may provide an important pathway for inducing hair-cell regeneration in the mammalian hearing organ.

Evidence for opening of hair-cell transducer channels after tip-link loss.

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 microM), amiloride (300 microM), 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.

The effect of explantation and neomycin on hair cells and supporting cells in organotypic cultures of the adult guinea-pig utricle.

Recent reports suggest that immature hair bundles are observed following aminoglycoside-induced hair-cell loss in the mammalian utricle in vitro as well as in vivo. It is therefore important to document the initial morphological changes associated with both culturing and aminoglycoside application so that degeneration can be clearly distinguished from regeneration. In this study, utricles from adult guinea pigs were maintained in culture for either 3 or 8 days, half being exposed to neomycin for days 2 and 3. They were then processed for microscopical examination and compared with control utricles from animals of the same age. The numbers of hair-cell and supporting-cell nuclei were counted and hair-cell morphology assessed. Bundles were classified as having either stepped (SHB) or unstepped (UHB) stereocilia, and their density determined. The numbers of hair-cell, but not supporting-cell, nuclei declined significantly compared with controls in both untreated and treated explants, the greatest reduction occurring 5 days after neomycin administration. The density of SHBs also declined but there was no significant change in UHB density, resulting in a residual population of hair bundles of more immature appearance in both untreated and treated utricles in vitro than in vivo. Although degenerative events such as hair-cell ejection from, or retraction into, the sensory epithelium were observed, no evidence of regeneration was found.

Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti.

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.

Immunocytochemical localization of a high-affinity glutamate-aspartate transporter, GLAST, in the rat and guinea-pig cochlea.

Glutamate transporters play an important role in the reuptake of glutamate after its release from glutamatergic synapses. Four such transporters have so far been cloned from the rat brain. One, the glutamate-aspartate transporter GLAST, has been detected in the mammalian cochlea, in which the principal afferent synapse of the auditory nerve, between the inner hair cells and neurites of type I spiral ganglion neurons, has been suggested to be glutamatergic. The distribution of GLAST was therefore investigated to provide clues to the handling of glutamate in the cochlea. This was studied using light and electron microscopic immunocytochemistry in rats and guinea pigs with antibodies raised against synthetic peptides based on the sequence for GLAST. Significant immunoreactivity was found in the myelin sheath formed by satellite cells surrounding the type I spiral ganglion neurons, and along the plasma membranes of supporting cells around the inner hair cells; other cells in both locations were only weakly labelled, if at all. The absence of substantial numbers of synapses in the spiral ganglion suggests that GLAST is unlikely to be associated with the uptake of synaptic glutamate after release in this region. Immunoreactivity associated with the inner hair cells is consistent with the utilization of glutamate at the afferent synapse.

Ultrastructural localization of cadherin in the adult guinea-pig organ of Corti.

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.

Kinematic analysis of shear displacement as a means for operating mechanotransduction channels in the contact region between adjacent stereocilia of mammalian cochlear hair cells.

In sensory hair cells of the cochlea, deflection of the stereociliary bundle results in direct mechanical gating of mechanoelectrical transduction channels, a function generally attributed to the tip link running between the tips of short stereocilia and the sides of adjacent taller ones. However, immunocytochemical experiments indicate that the channels may not be associated with the tip link but occur just below it in a region of contact between the stereocilia. To determine whether transduction channels in this location could be operated during physiologically appropriate deflections as effectively by shear displacement as if they were associated with the tip link, a two dimensional kinematic analysis of relative motion between stereocilia has been performed assuming contact between stereocilia is maintained during deflection. Bundle geometry and dimensions were determined from transmission electron micrographs of hair cells from several frequency locations between 0.27 and 13.00 kHz in the guinea-pig cochlea. The analysis indicates that for a 10 nm deflection of the tallest stereocilia of both inner and outer hair cells, i.e. within the range of the maximum sensitivity of mammalian hair bundles, the average shear displacement in the contact region would be 1.6 nm, but that it increases systematically towards higher frequency regions for outer hair cells. This displacement is comparable in magnitude to tip-link elongation for individual stereociliary pairs.

Immunoreactivity of sensory hair bundles of the guinea-pig cochlea to antibodies against elastin and keratan sulphate.

The stereociliary bundles of hair cells contain cross-linking extracellular filaments which have been suggested to play a role in mechanoelectrical transduction. To investigate the composition of these filaments, antibodies to the extracellular matrix molecules elastin and keratan sulphate have been used for light- and electron-microscopic immunocytochemistry of the guinea-pig organ of Corti. With the antibody to elastin, no immunoreactivity was found in hair bundles. This implies either that the epitope recognised by this antibody is not present in the links or that it is obscured. The antibody to keratan sulphate labelled the stereociliary bundles of both inner and outer hair cells but not supporting cells. The tips of the tallest stereocilia, especially on outer hair cells, the tips of the shorter stereocilia where the tip links attach to the stereociliary membrane, and the attachments of the lateral links, were labelled. This suggests that the links contain keratan sulphate proteoglycans, molecules which in other tissues are known to maintain structural integrity and fibrillar spacing, and to influence the microenvironment of the cell surface.