Spiro D. Comis

Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction

Hair cells of the guinea pig cochlea were preserved for electron microscopic examination by fixing in glutaraldehyde without the use of osmium. An extensive array of cross-links was seen between the stereocilia, by both scanning and transmission electron microscopy. The stereocilia were linked together laterally, particularly near their apical ends, by links running approximately at right angles to the long axis of the stereocilia. One set joined stereocilia of the same row, and another set joined stereocilia of the different rows, holding the tips of the shorter stereocilia in towards the longer stereocilia of the next row. In addition, the tip of each shorter stereocilium on the hair cell gave rise to a single, upwards-pointing link, which ran up to join the taller stereocilium of the next row. We suggest that distortion of this link would give rise to sensory transduction. On this basis, we are able to explain the V shape of the rows of stereocilia on outer hair cells. Within the rows, the three-dimensional arrangement of the stereocilia was different from that seen conventionally. Rather than standing parallel, the stereocilia of the different rows tapered in together at the tips, presumably held by the laterally-running cross-links. In addition, a membrane roughness, particularly pronounced in the region of the stereocilium which gives rise to the cross-links, was seen. However, the lateral and basal surface membranes of the hair cell, and the membranes of the internal organelles, had a more conventional appearance.

Morphology and cross-linkage of stereocilia in the guinea-pig labyrinth examined without the use of osmium as a fixative

SummaryHair cells of the guinea-pig cochlea and vestibular system were prepared for electron-microscopic examination by fixing in glutaraldehyde without the use of osmium. An extensive array of cross-links was seen between the apical ends of the stereocilia, by both scanning and transmission electron microscopy. Some cross-links ran laterally between stereocilia of the same row. Others ran laterally between the stereocilia of the different rows, holding the tips of the shorter stereocilia in towards the longer stereocilia of the next row. In addition, each tip on the shorter stereocilia gave rise to a single, upwards pointing link, which ran upwards to join the adjacent taller stereocilium of the next row. We suggest that distortion of this link might be involved in the mechanics or even the membrane biophysics of sensory transduction.With this method of preservation, all the apical surface membranes of the hair cells appeared rough, and contained dense granules. The roughness was greatest in the parts of the stereocilia to which the cross-links were attached. The mitochondrial and synaptic membranes of the hair cells appeared normal.

Vulnerability of tip links between stereocilia to acoustic trauma in the guinea pig

The cochleae of anaesthetized guinea pigs were prepared for scanning electron microscopy, immediately after exposure to an intense tone. Stereocilia on hair cells showing relatively small degrees of disruption were analyzed. If the bundles of stereocilia showed no or only a very slight degree of disorganization, the fine links emerging from the tips of the shorter stereocilia remained intact. If the stereocilia were separated more than a very little, the tip links between stereocilia were no longer visible. However, it was possible for tip links to remain intact in some parts of the hair bundle, while tip links in other, more disrupted parts, were lost. In outer hair cells, tip links did not seem any more vulnerable in one position than in another. In inner hair cells, it was commonly found that the tip links running between the tallest stereocilia and the next row of shorter stereocilia had broken, while the tip links running between the other shorter rows of stereocilia remained intact. The results suggest that tip links between stereocilia are preserved as long as the other links between the stereocilia and the cytoskeleton of the stereocilium remain intact. When the latter are damaged the tip links fracture. The results also suggest that, if the tip links are indeed involved in transduction, some degree of stimulus transduction can continue in damaged inner hair cells, albeit with a reduced sensitivity.

Osmium tetroxide postfixation in relation to the crosslinkage and spatial organization of stereocilia in the guinea-pig cochlea

SummaryGuinea-pig cochleae were fixed in glutaraldehyde, followed by short (30 s), intermediate (3 min) or long (24 h) periods of postfixation in 1% osmium tetroxide. The hair cells of the organ of Corti were examined by scanning electron microscopy. Following short periods of postfixation in osmium tetroxide, the preservation of crosslinks between the stereocilia could be enhanced, compared to the picture seen without postfixation. Moreover, in many cases the stereocilia parted slightly, revealing many lateral links between the stereocilia, which were otherwise hidden. The tip of each shorter stereocilium on a hair cell gave rise to a single, vertically pointing link, which ran upwards to the taller stereocilium of the adjacent row on the hair cell. It is suggested that distortion of such links is associated with sensory transduction. An extensive array of lateral links, connecting the stereocilia of the same and different rows on the hair cell, was also seen. In addition, the surface membranes of the stereocilia had a granular appearance. Following intermediate and long periods of postfixation in osmium tetroxide, crosslinks were rarely seen, and the stereocilia had smooth or wavy rather than granular surface membranes. Moreover, the spatial organization of the stereocilia, particularly on inner hair cells, was disrupted. In tissue postfixed for short periods, it was possible to map the directions of the vertically pointing links, and see the three-dimensional arrangement of the stereocilia. It was shown that the V-shape of the rows of stereocilia on outer hair cells, and the straight-line arrangement of the stereocilia on inner hair cells, were both appropriate for a maximal sensitivity of the hair cells to deflection of their stereocilia in a radial direction. It is suggested that the differences in the shape of the rows on inner and outer hair cells are derived from the different packing of the stereocilia on the two types of hair cell.

Further observations on the fine structure of tip links between stereocilia of the guinea pig cochlea.

Stereocilia of the guinea pig organ of Corti were examined by transmission electron microscopy, after fixation in glutaraldehyde and tannic acid, and postfixation and en bloc staining in osmium tetroxide, tannic acid, uranyl acetate, and phosphotungstic acid. Tip links were observed between the stereocilia. The links emerged from the tips of the shorter stereocilia in the hair bundle, running nearly at right angles to the cuticular plate, to join the side-wall of the adjacent taller stereocilium of the next row. The tip links had a fine filamentous core, approximately 6 nm in diameter. The core was surrounded by positively-staining amorphous material, which had a variable appearance from link to link. The central filament inserted into membrane specialisations at both its upper and lower ends. The results suggest that tip links have two components, and that the central filament, which has the same diameter as an actin filament, is suitable for transmitting stimulus-induced movements to the transducer channels of the stereocilium. The central filament would therefore concentrate the stimulus-induced forces onto a small area of cell membrane.

Possible Involvement of Nitric Oxide in the Sensorineural Hearing Loss of Bacterial Meningitis

Microperfusion of scala tympani with the NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), produced marked depression of the compound action potential (CAP) and cochlear microphonic (CM) together with severe and widespread morphological damage to hair cells and supporting cells in the organ of Corti. In addition, direct perfusion of N-methyl-D-aspartate (NMDA) into scala tympani, which probably induces excess stimulation of NMDA receptors within the cochlea and which is known to lead to the release of NO, was found to elicit similar electrophysiological and structural lesions in the cochlea. Pre-perfusion of scala tympani with L-methyl arginine (L-MA), which inhibits the release of NO, or superoxide dismutase (SOD), an O2-scavenger, conferred marked protection upon the cochlea from the lesions caused by NO donors. These observations indicate that enhanced NO production is likely to be an important factor responsible for pathological insult of the cochlea. The possibility is discussed that this factor is involved in the chain of events leading to hearing loss caused by bacterial meningitis. Such hearing loss is a major sequela of bacterial meningitis in children.

Cytotoxic Effects on Hair Cells of Guinea Pig Cochlea Produced by Pneumolysin, the Thiol Activated Toxin of Streptococcus pneumoniae

The cytolytic toxin, pneumolysin, from the gram positive bacterium, Streptococcus pneumoniae, when perfused through the scala tympani of the guinea pig cochlea reduced the amplitude of both the compound action potential and the cochlear microphonic potential. When the surface of the organ of Corti was examined by scanning electron microscopy, both inner and outer hair cells and supporting cells were found to be damaged. Inner hair cells and outer hair cells of row 3 were the most susceptible to damage by pneumolysin, followed by row 2 and then by row 1 of the outer hair cells. Damage to hair cells included disruption and splaying of stereocilia, loss of stereocilia and complete dissolution of hair bundles. Apical surfaces of hair cells and supporting cells were torn, pitted and cratered with shrinkage and tearing of cell boundaries. Within the dose range perfused (0.05-1 micrograms/microliters in a 10 microliters aliquot), the magnitude of the physiological and anatomical lesions was concentration dependent. The cytotoxic effects of pneumolysin reported here may be clinically significant factors in deafness caused by meningitis and otitis media in humans.

Early morphological and chemical changes induced by cisplatin in the guinea pig organ of Corti

Guinea pigs were treated with a single dose of Cisplatin (5 mg IP). After 2-4 days the cochleas were prepared for morphological analysis by scanning electron microscopy and chemical analysis by X-ray dispersive microanalysis. Following Cisplatin, the bundles of stereocilia on the hair cells were found to be rough, disarrayed, fused, and finally absorbed. Significant increases were found in the levels of calcium, sulphur, and phosphorus in the abnormal hair cells. It is suggested that the high calcium levels might be due to the inhibition of enzymes which normally keep cytoplasmic calcium low, and that some of the changes in the stereocilia might be secondary to this.

Cross-links between stereocilia in the human organ of Corti.

Human cochleae were fixed in glutaraldehyde, without the use of osmium. Cross-links were seen between the stereocilia, similar to those we have previously reported for the guinea pig: first, stereocilia of the same row on each hair cell were joined by horizontally-running links; secondly, the shorter stereocilia had pointed tips, each giving rise to a single, vertically-pointing link, which ran upwards to join the adjacent taller stereocilium of the next row. We suggest that distortion of this link is involved in sensory transduction. The links were sparser than had been seen in the guinea pig which may be a reflection of the vulnerability of the links to non-optimal fixation, and the greater difficulty in producing good fixation in human specimens.

Action of Elastase, Collagenase and Other Enzymes upon Linkages between Stereocilia in the Guinea-pig Cochlea

Enzymes, which degrade elements of the extracellular environment, were studied for their actions upon stereocilia and their cross-linkages by scanning electron microscopy. Chondroitinase, hyaluronidase and keratanase, which attack carbohydrate moieties of the extracellular matrix, had little effect upon hair bundles. Collagenase and plasmin (fibrinolysin) also had only marginal effects. Elastase produced dramatic effects upon hair bundles. Both lateral and tip links were degraded resulting in separation and splaying of stereocilia. Many stereocilia showed no marked loss of rigidity, although some were bent or kinked. In general, inner hair cells were the most susceptible to elastase followed by row 3, row 2, row 1 of the outer hair cells. The proteolytic enzyme trypsin did not noticeably disrupt the hair bundles. Protease caused loss of rigidity and fracture of stereocilia resulting in considerable collapse of hair bundles. Crosslinkages between stereocilia were less noticeably degraded. These results indicate that both lateral and tip links of stereocilia comprise a proteinaceous moiety which could be elastin or some chemically related structure.