Hair Cell Motility Research Articles

High frequency radial movements of the reticular lamina induced by outer hair cell motility

Recently, it was shown in cochlear explants from the guinea pig cochlea that electrokinetic motile responses of outer hair cells can induce radial and transverse motion of the reticular lamina. Here we demonstrate, that the radial component of these motions can be measured up to high frequencies (15 kHz). Cochlear explants were taken from guinea pig inner ears and exposed to a sinusoidal electric field. A double photodiode was used as a linear position detector with high spatial and temporatl resolution to detect radial movements in the plane of the reticular lamina. The organ of Corti of the second, third and fourth cochlear turns was stimulated with frequencies of the electrical field between 0.5 Hz and 20 kHz. Sinusoidal movements of up to 15 kHz were recorded. At higher frequencies the signal-to-noise ratio became too small. The largest responses were measured at the three rows of outer hair cells. If the strength of the electrical field was 2 kV/m, into which the cochlear explants were placed, the amplitudes of outer hair cell movements were around 1 μm at 1 Hz and 10 nm at 10 kH. Uncoupling of the outer hair cells from the tunnel of Corti and from the inner hair cells decreased the oscillations of inner hair cells but did not affect outer hair cells. The movements showed frequency dependent amplitudes like a complex low-pass filter but no best frequency was observed.

On the frequency limit and phase of outer hair cell motility: effects of the membrane filter

Whole-cell voltage clamp and displacement-measuring photodiode techniques were used to study electrophysiological and mechanical properties of the guinea pig outer hair cell (OHC). OHCs demonstrate a voltage-mechanical response (V-M) function that can be fit by a two state Boltzmann relation, where the cell normally rests near the hyperpolarizing saturation region (-70 to -90 mV). The voltage at half-maximal length change (Vh) is depolarized relative to the resting potential, and this ensures that for symmetrical sinusoidal voltage stimulation about the resting potential, AC and DC mechanical responses will be generated. Analysis of OHC motility using pure tone voltage bursts from 11 to 3200 Hz demonstrates both AC and DC mechanical responses. By exploiting the frequency-dependent current-voltage phase separation that is characteristic of an RC-dominated system under voltage clamp, it is demonstrated that OHC motility follows the phase of AC transmembrane voltage and not that of current. For voltage stimulation across frequencies in the acoustic range, the motility cutoff frequency corresponds to the cutoff frequency of the imposed transmembrane voltage. Frequency cutoffs approaching 1 kHz have been measured but are clamp time constant limited. These observations are congruent with the voltage dependency hypothesis of OHC motility. In addition, the DC component of the mechanical response is shown to be frequency independent, but to decrease in magnitude disproportionately compared to the AC component as the magnitude of the driving voltage decreases. This is predicted from the form of the V-M function, whose level dependent DC nonlinearity is a consequence of the resting potential being displaced from Vh. The net effect is that the mechanical DC: AC ratio approaches zero for small AC voltages. Taken together, these findings question the ability of the OHC mechanical response to influence organ of Corti micromechanics at high acoustic frequencies where a tuned amplification of basilar membrane motion is hypothesized.

Actin-binding and microtubule-associated proteins in the organ of Corti

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.

Reversible inhibition of voltage-dependent outer hair cell motility and capacitance

Outer hair cells (OHC) from the organ of Corti are capable of fast voltage-induced length changes (Santos-Sacchi and Dilger, 1988), suggesting that an associated voltage sensor should reside in the OHC plasma membrane. Voltage-dependent mechanical responses and nonlinear charge movement of isolated OHCs from the guinea pig were analyzed using the whole-cell voltage-clamp technique. Ionic currents in the cells were blocked. Nonlinear voltage-dependent charge movement or, correspondingly, voltage-dependent capacitance was measured with step or AC analysis. OHC movements were measured either from video or using a differential photodiode technique. Maximum charge movements up to 2.5 pC were measured in OHCs from the low-frequency region of the cochlea. Both AC and step analyses indicated a peak nonlinear capacitance of 16-17 pF. The voltage dependence was fit to a Boltzmann relation with the step analysis indicating a maximum nonlinear capacitance at -23 mV step potential from a holding potential of about -120 mV, and AC analysis indicating a maximum at a holding potential near -40 mV. AC analysis probably provides a more accurate evaluation of voltage dependence. Measures of OHC motility magnitude versus voltage follow the nonlinear capacitance-voltage function obtained from AC measures. Treatment of the cells with gadolinium ions (0.5-1 mM) blocked OHC motility. This treatment also produced a shift of the nonlinear capacitance function along the voltage axis in the depolarizing direction, which can be explained by membrane surface charge screening. However, maximum capacitance was reduced as well and may correspond to the reduction or abolition of OHC motility in response to gadolinium treatment. Gadolinium effects were reversible. Nonlinear capacitance is not a function of membrane deformation due to length changes, since removal of OHC cytosol via the patch pipette abolished longitudinal movement but did not reduce nonlinear charge movement. It is interesting to note that the nonlinear capacitance will dynamically influence the time constant of the OHC during acoustically evoked receptor potential generation.

The role of outer hair cell motility in cochlear tuning

The mammalian cochlea's remarkable sensitivity and frequency selectivity are thought to be mediated by the mechanical feedback action of outer hair cells. New tools for measuring the movement of cochlear elements, and recent advances in modeling are increasing our knowledge of cochlear mechanics.

Frequency-specific position shift in the guinea pig organ of Corti

The organ of hearing is tuned as expressed both in the vibratory response of the cochlear partition [2, 12, 16] and in the resulting receptor potentials of the sensory cells [6, 13]. We now demonstrate a sharply tuned response, consisting of a position shift of the surface of the organ of Corti, occurring during the presentation of a tone. The magnitude of the position shift exceeds that of the vibratory response to the stimulus. The shift is most pronounced in the region of the outer hair cells, and its affected by an inhibitor of outer hair cell motility. We conclude that the response is induced by the action of the outer hair cells.

The nature of the generator of outer hair cell motility

Despite the lack of obvious substrates underlying the electrokinetic shape changes of mammalian outer hair cells (OHCs), much can be learned about the mechanism when cells are electrically and mechanically partitioned in a glass microchamber. The results of such studies employing photometric as well as video analysis support the notion that the cellular motor consists of a large number of small, independent elements restricted to the supranuclear region of the cell's cortex. The length changes measured in OHCs are highly nonlinear. They are subject to manipulation as well as vulnerable to overstimulation. Thus the mechanical response resembles the mechanism underlying sharp tuning in the intact cochlea. Interestingly, such nonlinearities can be shown to be inherent to each voltage‐sensing element. The whole‐cell movement is thus effectively the cumulative response of many such elements, each driven in parallel by the local transmembrane potential along the length of the cell.

Differential motile response of isolated inner and outer hair cells to stimulation by potassium and calcium ions

Inner and outer hair cells were mechanically isolated from the guinea pig cochlea and subjected to stimuli known to induce shape changes in outer hair cells. Depolarization by 70 mM KCl which causes osmotic swelling of outer hair cells also swelled inner hair cells by approximately 8% of their volume. The application of the calcium ionophore ionomycin which induces cortical contractions and elongation of outer hair cells, did not affect the shape of inner hair cells. Since ionomycin increased free intracellular calcium levels in both inner and outer hair cells, the results demonstrate that inner hair cells do not possess the mechanisms necessary for a contractile response to calcium. Thus, calcium is a specific regulator of outer hair cell motility making this mechanism a likely physiological modulator of a transduction feedback process.

Effects of the crossed acoustic reflex on distortion-product otoacoustic emissions in awake rabbits

Recent studies in anesthetized cats suggest that contralateral-sound stimulation acts to suppress ipsilateral neural responses via the medial olivocochlear-efferent system. Activation of this descending efferent pathway presumably influences ipsilateral outer hair cell motility and, thus, cochlear micromechanics, resulting in reduced input to auditory-nerve fibers. The principal aim of the present study was to determine if contralateral-sound stimuli influence the generation of ipsilateral distortion-product otoacoustic emissions, in the ears of awake rabbits. The results showed no effects of contralateral stimuli on these emissions that could not be attributed to the crossed acoustic middle-ear reflex. The findings further indicate that distortion-product otoacoustic emission amplitudes over a wide range of frequencies can be dramatically reduced when the middle-ear reflex is activated.

Comparative ultrastructure of subsurface cisternae in inner and outer hair cells of the guinea pig cochlea

The subsurface cisternal systems of outer hair cells (OHCs) from different cochlear regions have been compared with the subsurface cisternal system of inner hair cells. Three main observations have been made: (1) the number of cisternal layers, when there is more than one present, is reduced along the length of an individual outer hair cell; (2) basal outer hair cells may have only one fenestrated cisternal layer; and (3) the inner hair cells possess a lateral cistern and associated pillar and filament complexes which are very similar to those of some basal OHCs. These observations are discussed in relation to hypotheses regarding the role of these structures in hair cell motility.

Cytoplasmic actin and cochlear outer hair cell motility.

Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaining cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.

Ultrastructural correlates of hair cell motility

Nonstimulated as well as outer hair cells showing mechanical displacements to electrical and chemical stimulation were examined ultrastructurally to assess the lability of a rhodamine‐labeled actin structure extending between the cuticular plate and the peri‐nuclear region. Hair cells of varying lengths (20–80 μm) were stimulated and immediately fixed following mechanical displacement. All cells revealed a distinct non‐membrane bound complex in cross section, but were infrequently observed in longitudinal sections. The cross‐sectional area of the structure increased in successive ascending sections. This structure spirals apically in a nondirect manner, but eventually consolidates into a primary mass to become contiguous with the inner leaflet of the cells plasmalemma. A localized microtubule network was found to pass most prominantly in the thin basal aspect of the complex. The cross‐sectional orientation appears to optimize the visualization of this potentially important aspect of the outer hair cells anatomy.

Electrokinetic model of cochlear hair cell motility.

Recent experiments have shown that isolated outer hair cells of the cochlea can vibrate under the influence of a transcellular oscillating electric field. Since high voltages have been measured in the cochlea, this result might suggest a basis for electromechanical feedback. A mechanical model of the hair cell has been developed and adapted to test the electrokinetic theory of motility, a postulate of which is that cochlear voltage gradients act on charged proteins embedded in the cell membrane to deform the cell. From the model it was deduced that the amount of charge density required is within the physiologic range. The significant result is that the amplitude of cell elongation for a fixed voltage amplitude is virtually constant for frequency less than a certain cutoff. The value of this frequency depends on the various physical parameters of the system and especially on the spacing between cells. Power transfer to the basilar membrane appears to peak near the cutoff frequency, and the amount is not very dependent on cell length, but is highly dependent on cell spacing.

A fast motile response in guinea-pig outer hair cells: the cellular basis of the cochlear amplifier.

1. Outer hair cells from the cochlea of the guinea-pig were isolated and their motile properties studied in short-term culture by the whole-cell variant of the patch recording technique. 2. Cells elongated and shortened when subjected to voltage steps. Cells from both high- and low-frequency regions of the cochlea responded with an elongation when hyperpolarized and a shortening when depolarized. The longitudinal motion of the cell was measured by a differential photosensor capable of responding to motion frequencies 0-40 kHz. 3. Under voltage clamp the length change of the cell was graded with command voltage over a range +/- 2 microns (approximately 4% of the length) for cells from the apical turns of the cochlea. The mean sensitivity of the movement was 2.11 nm/pA injected current, or 19.8 nm/mV membrane polarization. 4. The kinetics of the cell length change during a voltage step were measured. Stimulated at their basal end, cells from the apical (low-frequency) cochlear turns responded with a latency of between 120 and 255 microseconds. The cells thereafter elongated exponentially by a process which could be characterized by three time constants, one with value 240 microseconds, and a second in the range 1.3-2.8 ms. A third time constant with a value 20-40 ms characterized a slower component which may represent osmotic changes. 5. Consistent with the linearity shown to voltage steps, sinusoidal stimulation of the cell generated movements which could be measured at frequencies above 1 kHz. The phase of the movement relative to the stimulus continued to grow with frequency, suggesting the presence of an absolute delay in the response of about 200 microseconds. 6. The electrically stimulated movements were insensitive to the ionic composition of the cell, manipulated by dialysis from the patch pipette. The responses occurred when the major cation was K+ or Na+ in the pipette. Loading the cell with ATP-free solutions or calcium buffers did not inhibit the response. 7. It is concluded that interaction between actin and myosin, although present in the cell, is unlikely to account for the cell motility. Instead, it is proposed that outer hair cell motility is associated with structures in the cell cortex. The implications for cochlear mechanics of such force generation in outer hair cells are discussed.

Measurements of in vitro outer hair cell motility in the mammalian cochlea

The sensory receptors of albino guinea pigs were dissociated and maintained in high sodium culture media. Outer hair cells were examined in response to current stimulation and cholinomimetic compounds. Two types (I and II) of movement have been detected. Small type I responses were seen when stimulated by electrodes placed in the medium along the long axis of the cell similar to those observed by Brownell (SEM/III, 1984). Large type I responses at higher current densities were seen by the suction electrode technique. These motile events appear to be graded in response and restricted to the supranuclear region. A video camera and phototransister were utilized with an inverted microscope. A signal averaging system allowed the recording of the displacement function of the cell. Frequency responses of the cells were derived and the input‐output function modeling the movement was determined. Type II acetylcholine or carbachol‐induced movements were seen in a proportion of the cells examined. These movements were slow sustained contractions which appear to be reversible when the compound is washed out of the medium. We found the outer hair cell to be exquisitely sensitive to osmotic fluctuation. Further evidence confirming efferent stimulation is presently being sought.

A model of the effect of outer hair cell motility on cochlear vibrations.

A model of cochlear function is presented in which deformation forces within outer hair cells are assumed to occur in synchronized response to generator potentials. Assuming a 90 degree phase lag between the generator potentials and the deformation forces, it is shown that the forces act to reduce cochlear-partition damping and thus increase frequency selectivity. A number of other experimentally observed phenomena, such as the effects of efferent-fiber stimulation and electrical polarization, can also be accounted for with this model.

Motile responses in outer hair cells.

Motile responses of cochlear hair cells open new perspectives for the understanding of cochlear hearing mechanisms and hearing disorders located in hair cells. Direct visualization of hair cell motility was achieved by a method for the study of living isolated mammalian outer hair cells (OHCs) which has overcome some of the complexities in dealing with the heterogeneous organ of Corti. Electrophysiological giga-seal whole-cell recordings of single OHC prepared by this approach had revealed negative cell potentials ranging from -32 mV to -70 mV (Gitter et al. (1986) Oto-Rhino-Laryngol. in press). Elucidation of HC motility has come from two lines of experiments. One follows from the observation that exposure of the lateral and basal membrane parts of living OHCs to increasing bath K+ concentrations resulted in a sustained reversible depolarization of the cell. Here, we report that by depolarization of the cell membrane in the presence of 25-125 mM K+/Cl- a sustained contraction of OHC was induced. This was followed by relaxation in the presence of artificial perilymph containing 5.4 mM K+/Cl-. By alternating these procedures OHCs were made to undergo as many as five cycles of contraction and relaxation. External Ca2+ was not required for the initial contraction but was essential for relaxation. Following repeated contraction/relaxation cycles the cytoplasm of individual OHCs exhibited a filamentous network, correlating with a new infracuticular anti-actin binding capacity. The second series of experiments originates in the observation that permeabilized OHCs contracted in the presence of ATP. No response was seen in the presence of control nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)