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C M Hackney

Publications and source records attributed to C M Hackney.

At least 19 recordsLinked to original sources
PubMed2001

Clues to the cochlear amplifier from the turtle ear.

Sound stimuli are detected in the cochlea by vibration of hair bundles on sensory hair cells, which activates mechanotransducer ion channels and generates an electrical signal. Remarkably, the process can also work in reverse with additional force being produced by the ion channels as they open and close, evoking active movements of the hair bundle. These movements could supplement the energy of the sound stimuli but to be effective they would need to be very fast. New measurements in the turtle ear have shown that such active bundle movements occur with delays of less than a millisecond, and are triggered by the entry of Ca(2+) into the cell via the mechanotransducer channel. Furthermore, their speed depends on the frequency to which the hair cell is most sensitive, suggesting that such movements could be important in cochlear amplification and frequency discrimination.

Animals↗
PubMed2000

An immunogold investigation of the relationship between the amino acids GABA and glycine and their transporters in terminals in the guinea-pig anteroventral cochlear nucleus.

The majority of terminals contacting spherical bushy cell bodies in the guinea-pig anteroventral cochlear nucleus contain GABA, glycine or both (colocalizing). Double labeling with antibodies to each amino acid and the plasma membrane transporter for the other was performed using different sizes of gold particles. The transporter for GABA occurs in the plasma membranes of some terminals containing glycine and vice versa suggesting that colocalizing terminals can retrieve both amino acids.

Amino Acid Transport Systems, Neutral↗
PubMed2000

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.

ATP-Binding Cassette Transporters↗
PubMed2000

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

Animals↗
PubMed1999

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.

Acoustic Stimulation↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

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.

Animals↗
PubMed1996

The binding site on cochlear stereocilia for antisera raised against renal Na+ channels is blocked by amiloride and dihydrostreptomycin.

The mechanoelectrical transduction channels on hair cells have been suggested to be operated by tip links that are stretched when the hair bundle is deflected in the direction of the tallest row of stereocilia. Localising these channels is therefore an important test of this hypothesis. The transduction channels are known to be amiloride-sensitive and immunogold labelling with antibodies raised against the amiloride-sensitive epithelial Na+ channel from kidney (alpha NaCh), has suggested that sites with similar characteristics are located in the region where the tips of the shorter stereocilia appear to come into contact with the sides of the adjacent taller stereocilia rather than being associated directly with the tip links. Now, further immunocytochemical experiments have been performed to determine if amiloride and dihydrostreptomycin, both of which can block transduction, can affect this labelling. Immunofluorescent labelling of the stereocilia is obtained when surface preparations of the organ of Corti are fixed and incubated with alpha NaCh followed by an appropriate secondary antibody. This labelling is abolished by trypsinization prior to fixation but retained if the tissue is pretreated with amiloride and then trypsinized in its presence. Because amiloride is known to protect amiloride-binding sites from degradation by trypsin, these results suggest that alpha NaCh is revealing amiloride-binding sites on the stereocilia. Similarly, immunofluorescent labelling of the stereocilia is abolished if cochlear tissue is pretreated with dihydrostreptomycin (DHS) and fixed in its presence prior to incubation with alpha NaCh. Quantitative analysis of colloidal gold labelling using transmission electron microscopy shows that DHS treatment produces a significant reduction in the number of gold particles on stereocilia, especially in the region of contact between them. These results suggest that anti-Na+ recognises a site with characteristics similar to the mechanoelectrical transduction channels.

Amiloride↗
PubMed1996

Immunocytochemical evidence that glutamate is a neurotransmitter in the cochlear nerve: a quantitative study in the guinea-pig anteroventral cochlear nucleus.

The large so-called type I afferents of the cochlear nerve carry the majority of the auditory input from the cochlea to the cochlear nuclei in the brainstem. These fibres are excitatory and previous studies have suggested they may use glutamate as their neurotransmitter. In the present investigation therefore, antibodies to glutamate and to the glutamate precursor, glutamine, were applied to resin sections of perfusion-fixed brains and of in vitro brain slices subjected to depolarizing levels of potassium before fixation to study glutamate handling and synaptic release. Ultrathin sections were labelled by the immunogold technique, and the immunoreactivity was quantified by recording the density of gold particles over the various tissue profiles. Non-primary, presumably inhibitory, terminals and glial processes were used as reference structures. The cochlear primary terminals proved to be strongly immunoreactive for glutamate. The density of glutamate labelling was higher in primary terminals than in non-primary ones, and lowest in glial processes. The ratio between the mean glutamate and glutamine labelling densities was also higher in primary terminals than in non-primary ones, and lowest in glial processes in each case. In the primary terminals, the glutamate immunoreactivity was higher over vesicle-containing regions than over vesicle-free regions, whilst glutamine was evenly distributed throughout. The in vitro brain slices showed a potassium-induced, partly calcium-dependent depletion of glutamate from the primary terminals but not from the non-primary ones. These observations strongly support the conclusion that glutamate is a neurotransmitter of type I cochlear afferents.

Afferent Pathways↗
PubMed1995

Aluminium, beta-amyloid and non-enzymatic glycosylation.

The non-enzymatic glycosylation of beta-amyloid is implicated in the aetiology of Alzheimer's disease. However, controversy surrounds the nature of any involvement and a potential mechanism has not been fully elucidated. We present evidence of an aluminium-induced aggregation of the A beta P(25-35) peptide and speculate that the mechanism of formation of our ordered beta-amyloid aggregates might involve non-enzymatic glycosylation and/or site-specific crosslinking of beta-amyloid fibrils by atomic aluminium.

Aluminum↗
PubMed1995

Mechanotransduction in vertebrate hair cells: structure and function of the stereociliary bundle.

The mechanosensitive hair cells of the vertebrate acousticolateralis system have an apical bundle of stereocilia, deflections of which control the opening of mechano-electrical transduction channels and thus generate receptor potentials in the cell below. This review describes current theories of hair cell function in the light of recent immunocytochemical and ultrastructural findings; in particular, the location and operation of the transduction channels are considered. The most widely accepted hypothesis of mechanotransduction by hair cells is that fine extracellular links that run between the tips of shorter stereocilia and the sides of taller ones operate the transduction channels. However, the fact that the transduction channels are amiloride sensitive has led to labeling experiments using antibodies to the amiloride-sensitive epithelial Na+ channel from kidney which suggest that the mechanotransduction channels may not be directly associated with the tip links. Instead, they appear to be located near a junctionlike structure at the point of contact between the shorter and taller stereocilia. The implications of these findings for the tip link hypothesis are discussed.

Animals↗
PubMed1993

The functional morphology of stereociliary bundles on turtle cochlear hair cells.

The stereociliary bundles of hair cells from the basilar papilla of the red-eared turtle were examined with transmission and high resolution scanning electron microscopy to provide a description of their morphology, orientation and inter-ciliary connections for comparison with physiological observations. Bundles on hair cells in the basilar membrane region are of a uniform shape and orientation, but bundles on the apical and basal limbus are distinct in having elongated kinocilia more than twice the length of the tallest stereocilia. Bundles in the basilar membrane region show a roughly two-fold increase in height from 5 to 9 microns from base to apex. Electrical recordings from isolated hair cells indicate that the bundle height is inversely proportional to the cell's characteristic frequency. It is argued that the change in dimensions is insufficient to contribute significantly to the cochlea's frequency selectivity. The cytoplasm adjacent to the kinocilium is filled with microtubules and large vesicles, and there are coated pits in the apical membrane which, it is suggested, may be indicative of rapid turnover of the membrane in this region.

Animals↗