SUMMARY1. The mechanical behaviour of the ciliary bundles of hair cells in the turtle cochlea was examined by deflecting them with flexible glass fibres ofknown compliance during simultaneous intracellular recording of the cell's membrane potential.2. Bundle motion was monitored through the attached fibre partially occluding a light beam incident on a photodiode array. The change in photocurrent was assumed to be proportional to bundle displacement.3. For deflexions of 1-100 nm towards the kinocilium, the stiffness of the ciliary bundles was estimated as about 6 x 10-4 N/m, with the fibre attached to the top of the bundle.4. When the fibre was placed at different positions up the bundle, the stiffness decreased approximately as the inverse square of the distance from the ciliary base. This suggests that the bundles rotate about an axis close to the apical pole of the cell and have a rotational stiffness of about 2 x 10-14 N. m/rad. 5.Step displacements of the fixed end of the flexible fibre caused the hair cell's membrane potential to execute damped oscillations; the frequency of the oscillations in different cells ranged from 20 to 320 Hz. Displacements towards the kinocilium always produced membrane depolarization.6. The amplitude of the initial oscillation increased with displacements up to 100 nm and then saturated. For small displacements of a few nanometres, the hair cell's mechanoelectrical sensitivity was estimated as about 0-2 mV/nm. 7. Force steps delivered by the flexible fibre caused the bundle position to undergo damped oscillations in synchrony with the receptor potential. The mechanical oscillations could be abolished with large depolarizing currents that attenuated the receptor potential.8. When placed against a bundle, a fibre's spontaneous motion increased and became quasi-sinusoidal with an amplitude several times that expected from the compliance of the system. It is suggested that the hair bundle drives the fibre.9. We conclude that turtle cochlear hair cells contain an active force generating mechanism.

SUMMARY1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under wholecell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber.2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz.3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200-400 pA were observed for deflections of the tip of the bundle of 0 5 ,tm, equivalent to rotating the bundle by about 5 deg.4. In response to a step deflection of the bundle, the current developed with a time constant (about 0 4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 'C drop in temperature.5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3-5 ms. This adaptation was associated with a shift of the current-displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+.6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mm of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels.7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement.8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.NIS 77W0

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.

SUMMARY1. Mechano-electrical transducer currents evoked by deflections of the hair bundle were recorded in turtle isolated hair cells under whole-cell voltage clamp. The outcome of perfusing with solutions of reduced Ca2+ concentration was investigated.2. The transducer current was roughly doubled by lowering the concentration of divalent cations from normal (2-2 mM-Mg2+, 2-8 mM-Ca2+) to 0 Mg2+, 0 5 mM-Ca2+. No significant effects on the current's kinetics or reversal potential, or on the currentdisplacement relationship, were noted.3. If the Ca2+ concentration was lowered to 50 ,UM (with no Mg2+), there was about a threefold increase in the maximum current but other changes, including loss of adaptation and a decreased slope and negative shift in the current-displacement relationship, were also observed. As a result, more than half the peak transducer current became activated at the resting position of the hair bundle compared to about a tenth in the control solution.4. The extra changes manifest during perfusion with 50 /LM-Ca2+ had also been seen when the cell was held at positive potentials near the Ca2+ equilibrium potential. This supports the view that some consequences of reduced external Ca2+ stem from a decline in its intracellular concentration.5. With 20,tM-Ca2+, a standing inward current developed and the cell became unresponsive to mechanical stimuli, which may be explained by the transducer channels being fully activated at the resting position of the bundle.6. The results are interpreted in terms of a dual action of Ca2+: an external block of the transducer channel which reduces the maximum current, and an intracellular effect on the position and slope of the current-displacement relationship; the latter effect can be modelled by internal Ca2+ stabilizing one of the closed states of the channel.7. During perfusion with 1 gM-Ca2 , the holding current transiently increased but then returned to near its control level. There was a concomitant irreversible loss of sensitivity to hair bundle displacements which we suggest is due to rupture of the mechanical linkages to the transducer channel.

Outer hair cells are centrally involved in the amplification and frequency tuning of the mammalian cochlea, but evidence about their transducing properties in animals with fully developed hearing is lacking. Here we describe measurements of mechanoelectrical transducer currents in outer hair cells of rats between postnatal days 5 and 18, before and after the onset of hearing. Deflection of hair bundles using a new rapid piezoelectric stimulator evoked transducer currents with ultra-fast activation and adaptation kinetics. Fast adaptation resembled the same process in turtle hair cells, where it is regulated by changes in stereociliary calcium. It is argued that sub-millisecond transducer adaptation can operate in outer hair cells under the ionic, driving force and temperature conditions that prevail in the intact mammalian cochlea.