J.O. Pickles

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.

The effects of moderate and low levels of acoustic overstimulation on stereocilia and their tip links in the guinea pig

Guinea pigs were exposed to pure tones of 10 kHz at intensities between 98 and 115 dB SPL for 5-30 min, to produce varying degrees of acoustic trauma. Changes in auditory thresholds were measured electrophysiologically, and the animals were immediately fixed for scanning electron microscopy. Correlation between morphological changes to the hair bundle and losses in threshold, showed that with the smallest degrees of trauma (98 dB SPL for 15 min, mean maximum threshold loss of 22 dB), damage was confined to a small stretch of inner hair cells (IHC), with only subtle changes to the stereocilia of the outer hair cells (OHC). At exposure intensities greater than 102 dB SPL (duration: 15 min) the IHC stereocilia in the centre of the lesion were always substantially disarrayed. Substantial damage to the OHC bundles was seen only with exposures above 110 dB SPL (duration: > or = 5 min), producing threshold losses of 50 dB or more. Tip links were lost only where the stereocilia were disarrayed. It is concluded that the tip links are not the most vulnerable components of the cochlear hair cell, but that relatively low levels of acoustic stimulation can cause significant damage to the stereociliary bundle of the IHCs.

Frequency threshold curves and simultaneous masking functions in single fibres of the guinea pig auditory nerve.

Tuning curves for simultaneous masking were measured electrophysiologically, in single fibres of the guinea pig auditory nerve. The masking paradigm used was an analogy of that used in the determination of psychophysical tuning curves in man. The resulting tuning curves ran nearly parallel to the corresponding neural frequency threshold curve, over all except the high-frequency portion of the tuning curve. There, the masking functions had a shallower slope than the excitatory frequency threshold curve. The frequency at which the slope became shallower had a close and consistent dependence on fibre characteristic frequency, reaching a value of 1.2 times fibre characteristic frequency in high-frequency fibres. Analysis of firing rates during the threshold determination gave information about the mechanism of the masking, and the results were supported by theoretical analysis. The results give information useful for the interpretation of psychophysical tuning curves, determined by simultaneous masking, in man.

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.

The effect of chronic application of kanamycin on stereocilia and their tip links in hair cells of the guinea pig cochlea

Albino guinea pigs were treated with kanamycin (400 mg/kg i.p.) daily for 10 days. After a 2-week recovery period their cochleae were analyzed by scanning electron microscopy. Attention was paid to those outer hair cells which had been less severely damaged. Stereocilia of the outer hair cells were often constricted at the root, and in some cases had become detached at the root. In stereocilia showing any degree of abnormality, the tip links were usually missing. However, in a few exceptional cases, tip links could remain on stereocilia showing other abnormalities, such as constriction at the root. Where hair cells were otherwise apparently unaffected, a much higher proportion of tip links remained, even on cells situated in an area of extensive hair cell loss. The results give further information on the process of kanamycin poisoning. They also suggest that substantial loss of tip links, and therefore perhaps of transduction, is one of the preliminary consequences of kanamycin poisoning.