Guinea Pig Outer Hair Cells Research Articles

Acetylcholine activates a K + conductance permeable to Cs + in guinea pig outer hair cells

Acetylcholine (ACh), the major neurotransmitter released by efferent nerve fibers in the cochlea, has been shown to activate a Ca 2+-dependent K + conductance in outer hair cells (OHCs). Previously we reported that this ACh operated conductance is permeable to Cs +. The purpose of the present study was to characterize further this Cs +-permeable channel and its dependency on Ca 2+ using isolated OHCs and the patch clamp technique in the whole cell configuration. The changes in the ACh response were examined when Cs +, Ba 2+, Cd 2+, N-methyl-D-glucamine (NMG +) and tetraethylammonium (TEA +) were placed in the external or internal solutions. Cs + substituted for K + in carrying the ACh-evoked Ca 2+-dependent K + current. When NMG +TEA + was substituted for internal K + ACh-evoked an inward and an outward current, and Cs + substituted for external K + blocked the outward but not the inward current evoked by ACh suggesting it was carried by K +. In the NMG +TEA + condition, when the cell was held at different V h values for an extended period of time, the ACh-induced K + current rectified. In Ba 2+ (3 mM) with zero Ca 2+ ACh failed to induce any detectable current and the ACh response slowly recovered from the Ba 2+ block, suggesting a block at an intracellular site. Cd 2+ (1 mM) readily and reversibly blocked ACh-induced currents even when carried by Cs +. This data suggests that ACh opens a channel selective for K +, conductive to Cs + and dependent on Ca 2+.

Physiologically silent sodium channels in mammalian outer hair cells

1. Voltage-dependent properties of isolated guinea pig outer hair cells (OHCs) were investigated using whole-cell recording. An inward current was detected in approximately 10% of the cells. This inward current was identified as belonging to the voltage-activated sodium current family on the basis of its high sensitivity to tetrodotoxin and the effect of substitution of impermeant ions. Although this is the first report of a sodium current in the mammalian cochlea, it differs from the classical neuronal sodium current by having a variable magnitude from cell to cell and an inactivation that is shifted to hyperpolarized potentials. The sensory processing role of hair cells in general and outer hair cells in particular could be disrupted by the presence of a regenerative voltage-dependent current. The functional role of the OHC sodium channels is puzzling, particularly as they may be silent in vivo.

F-actin, tubulin and spectrin in the organ of Corti: Comparative distribution in different cell types and mammalian species

Laser scanning confocal microscopy was used to determine the distribution of actin, spectrin and tubulin in whole mounts of the organ of Corti of guinea pig, monkey, rat and chinchilla. Actin, spectrin and tubulin were localized in all cell types in the auditory epithelium. No specialized cytoskeletal organization of tubulin was detected in the cytoplasmic domain of hair cells. The only specialized organization of actin and spectrin in the cytoplasmic domain was the infra-cuticular network, found exclusively in apical guinea pig outer hair cells. In contrast, the lateral wall of inner and outer hair cells contained a homogenous distribution of label specific for actin and spectrin. The label intensity was similar in the base and the apex of the cochlea. These results indicate that the distribution of spectrin and actin in the auditory epithelium is similar to that in other epithelial cells, suggesting that actin and spectrin participate in the formation of cellular shape and possibly in docking molecules to the membrane.

Frequency response of mature guinea-pig outer hair cells to stereociliary displacement

Outer hair cells (OHC) were isolated from the apical two turns of the guinea-pig cochlea and their hair-bundle stimulated mechanically by a glass probe. In accordance with in vivo data ( Dallos, 1985), the resting membrane potential was typically −64 mV ( N = 200). The maximum amplitudes of the receptor potentials were between 0.4 and 5.2 mV peak-to-peak, with mean of 1.5 mV ± 0.9 mV ( N = 81). The sensitivity was 0.015 mV/nm or 2 mV/deg. The frequency response of the receptor potential followed a first order low-pass filter characteristic with a corner frequency of about 63 Hz. For frequencies up to at least 1.6 kHz, the frequency response of mechanoelectrical transduction was dominated by the electrical input impedance of the cell. The presence of a single time constant in the voltage response to stereociliary deflection implies that the frequency response of mechanoelectrical transduction far exceeds that of the electrical input impedance of the cell; its time constant must be faster than 100 μs. Under in vivo conditions, OHC should be capable of providing a sufficiently large receptor potential to supply enough energy for electromechanical feedback.

Specific glutathione-SH inhibition of toxic effects of metabolized gentamicin on isolated guinea pig hair cells.

The present investigation has shown that only metabolized gentamicin (mG) but not native gentamicin (G) is cytotoxic for isolated guinea pig outer hair cells (OHC). Using FURA-2 fluorescence, both G and mG were found to reduce intracellular free Ca2+ concentrations in unstimulated OHC and inhibit increases in intracellular Ca2+ concentrations during K(+)-induced depolarization. Glutathione-SH (GSH), a detoxificating agent, did not interfere with G and mG effects on intracellular Ca2+ concentration. In non-stimulated OHC, mG but not G induced pathological OHC depolarization, indicating the opening of transduction channels to allow influx of K+ ions. GSH completely inhibited the lytic effect of mG. Electrophysiological investigations also revealed that GSH probably inhibits mG-induced pathological opening of transduction channels. These results suggest that GSH selectively inhibits mG-specific toxic effects on the guinea pig OHC, possibly by enzymatic detoxification.

Charge displacement induced by rapid stretch in the basolateral membrane of the guinea-pig outer hair cell.

The properties of the basolateral membrane of cochlear outer hair cells were studied under whole-cell patch clamp to measure currents and capacitance changes associated with mechanical deformation. Stretching the membrane of outer hair cells along the cell axis generated a transient inward current, and subsequent relaxation of the membrane produced a similar transient outward current. These mechanically activated currents were velocity dependent with a mean sensitivity of 29 pA s mm-1. Unlike ionic currents, these currents did not reverse, but reached a peak magnitude at -33 mV. Stretching the cell also resulted in a measurable capacitance decrease of 0.3-1.1 pF microns-1. These results suggest that membrane stretch can induce a rapid charge movement resulting from the reversal of the electromechanical transduction process in outer hair cells.

Tuned phasic and tonic motile responses of isolated outer hair cells to direct mechanical stimulation of the cell body

Guinea pig outer hair cells (OHCs) isolated from the two apical turns of the cochlea and firmly attached to a suction pipette, were subjected to the stimulus of the near-field (particle) displacements of a calibrated oscillating fluid jet aimed at the lateral cell walls. The longitudinal length changes of the OHCs in response to stimulation, in a direction orthogonal to that of the fluid jet, were recorded by a photodiode array. The response had two components; a phasic length change which followed the frequency of the particle displacement of the jet cycle by cycle, and a tonic length change which took several milliseconds to develop depending on the magnitude of the mechanical stimulus. When the hair cell changed length the lateral walls of the OHC moved in antiphase, moving apart during shortening and together during lengthening. With increased stimulus level the phasic response grew in proportion to the stimulus magnitude and began to saturate at the highest stimulus levels, while the tonic response grew in proportion to the square of the stimulus magnitude. Both the direction of the tonic length change and the phase of the phasic component could alter with the level of stimulation. Isolevel and isoresponse -frequency functions of both the tonic and phasic length changes revealed that both response types were tuned to similar resonant frequencies (RF) between 150 and 2500 Hz. The phase of the phasic length change began to lag at frequencies just below the RF, lagged by about 90° at the RF and lagged by a further 90° at frequencies above RF. The frequency response properties of the OHCs closely corresponded to those of a damped, forced, mechanical resonance. The tonic response disappeared and the phasic response was reduced at low-levels as a consequence of intense mechanical stimulation and with time.

Comparative electrophysiological properties of guinea pig ( Cavia cobaya) outer hair cells and frog ( Rana pipiens) semicircular canal hair cells

The morphological characteristics of isolated guinea-pig outer hair cells (OHCs) and frog semicircular canal hair cells (SCCHCs) were reviewed. Active and passive electrophysiological properties of OHCs and SCCHCs were compared utilizing the whole cell variant of the patch clamp technique. Results lead to the conclusion that both hair cell types are similarly viable. SCCHCs were dominated by inactivating outward conductances and were strongly inwardly rectified. In contrast, OHCs demonstrated little inactivation or rectification. The differences in electrophysiological properties between the two cell types may reflect the separate functions these cells have in the ear.

Voltage-dependent block by neomycin of the ATP-induced whole cell current of guinea-pig outer hair cells.

1. The effects of externally applied ATP and neomycin on whole cell currents of isolated guinea pig cochlear outer hair cells (OHCs) were studied using the whole cell voltage-clamp technique. In OCHs held at -70 mV, ATP activated a large inward current. In the presence of neomycin, the ATP-induced whole cell current activated along a relatively unaltered time course, but the current then decreased to a reduced steady level. The neomycin inhibition of the ATP-induced current was dose dependent. The half-inhibitory concentration (IC50) of neomycin measured at steady state was estimated to be 90 microM. 2. Neomycin inhibition of the ATP response could not be reversed by increasing the concentration of ATP, indicating that the effect was noncompetitive. The inhibition was voltage dependent and was greatly reduced when OHCs were held at positive potentials. 3. Cells treated with 100 microM ATP gave maximal current responses. Addition of neomycin substantially increased membrane current noise of the 100 microM ATP responses. When neomycin concentration was varied from 10 to 500 microM, the current noise level peaked between 50 and 100 microM. The noise increase was observed at negative holding potentials but not at positive potentials. 4. The neomycin-induced whole cell current noise was used to estimate the size of the underlying elementary current. The ATP-induced single channel current of OHCs at -70 mV was estimated to be approximately 0.3 pA. The number of ATP-activated channels in a single OHC was estimated to be in the range of a few thousand. 5. The characteristics of the neomycin inhibition of ATP-induced currents were consistent with an open channel blocking mechanism. Analysis of the voltage dependence of the steady state neomycin inhibition suggested a neomycin binding site at an electrical distance of 0.3 from the extracellular side.

The location and mechanism of electromotility in guinea pig outer hair cells.

1. The microchamber method was used to examine the motile responses of isolated guinea pig outer hair cells to electrical stimulation. In the microchamber method, an isolated cell is drawn partway into a suction pipette and stimulated transcellularly. The relative position of the cell in the microchamber is referred to as the exclusion fraction. 2. The length changes of the included and excluded segments were compared for constant sinusoidal stimulus amplitude as functions of the exclusion fraction. Both included and excluded segments showed maximal responses when the cell was excluded approximately halfway. Both segments showed smaller or absent responses when the cell was almost fully excluded or almost fully included. 3. When the cell was near to, but not at, the maximum exclusion, the included segment response amplitude was zero, whereas the excluded segment response amplitude was nonzero. In contrast, when the cell was nearly fully included, the excluded segment response amplitude was zero, but the included segment response amplitude was still detectable. A simple model of outer hair cell motility based on these results suggests that the cell has finite-resistance terminations and that the motors are restricted to a region above the nucleus and below its ciliated apex (cuticular plate). 4. The function describing length change as a function of command voltage was measured for each segment as the exclusion fraction was varied. The functions were similar at midrange exclusions (i.e., when the segments were about equal length), showing nonlinearity and saturability. The functions were strikingly different when the segment lengths were different. The effects of exclusion on the voltage to length-change functions suggested that the nonlinearity and saturability are local properties of the motility mechanism. 5. The diameter changes of both segments were examined. The segment diameter changes were always antiphasic to the length changes. This finding implies that the motility mechanism has an active antiphasic diameter component. The diameter change amplitude was a monotonically increasing function of exclusion for the included segment, and a decreasing function for the excluded segment. 6. The voltage to length-change and voltage to diameter-change functions were measured for the same cell and exclusion fraction. The voltage to diameter-change function was smaller in amplitude than the voltage to length-change function. The functions were of opposite polarity to each other, but were otherwise similar in character. Thus it is likely that the same motor mechanism is responsible for both axial and diameter deformations.

Hyposmotic activation hyperpolarizes outer hair cells of guinea pig cochlea

The electrophysiological responses of isolated guinea pig outer hair cells (OHCs) to hyposmotic activation were studied using the whole-cell patch-clamp technique. The cell swelling by hyposmotic activation hyperpolarized OHCs by 6.6 ± 2.3 mV from the resting membrane potential of −58.5 ± 5.9 mV ( n = 48). This hyperpolarization was associated with an outward current ( 97.7 ± 22.2, pA, n = 15). The hyperpolarization was inhibited by 300 μM quinine, 5 mN Ba 2+ and increasing the extracellular K + to 30 mM from 5 mM. In the absence of extracellular Ca 2+ (1 mM EGTA), the hyperpolarization during hyposmotic activation was also abolished while the following depolarization was preserved. 50 μM GdCl 3, which is known to block strecch-activated non-specific cation channels, inhibited the hyperpolarization reversibly. 50 μM GdCl 3 also inhibited [Ca 2+] i increase during hyposmotic activation as shown by the calcium-sensitive dye fura-2. Simultaneously, the [Ca 2+] i increase and the hyperpolarization during hyposmotic activation could be observed using the combined method of whole-cell patch clamp and fura-2 technique. It is concluded that the cell swelling by hyposmotic activation may activate the stretch-activated non-specific cation channels in the OHCs which allow a Ca 2+ influx. In turn, this [Ca 2+] i increase leads to an activation of the Ca 2+-activated K + channels at the basolateral membrane of OHCs which results finally in a reversible hyperpolarization of OHCs by K + efflux.

Acetylcholine increases intracellular Ca 2+ concentration and hyperpolarizes the guinea-pig outer hair cell

Extracellularly applied acetylcholine (ACh) induced outward currents in isolated outer hair cells of a guinea-pig cochlea. The ACh induced current was carried by K + ions. The current amplitude was ACh dose dependent with a K D of 12 μM. The ACh induced outward current was reversibly blocked by extracellularly applied atropine (1 μM), d-tubocurarine (d-TC, 1 μM), apamin (1 μM) and strychnine (0.1–10 μM). D-TC (10 μM) not only blocked the ACh induced outward current, but also reduced the amplitude of depolarization induced outward current. ACh induced a rise of intracellular Ca 2+ concentration ([Ca 2+]i). D-TC (10 μM) reduced but did not totally block the increase of [Ca 2+]i. In a low Ca 2+ (0.1 mM) extracellular medium, the amplitude of ACh induced current was reduced rapidly and was recovered gradually to the normal level after the extracellular Ca 2+ concentration was resumed. It is probable that ACh hyperpolarizes the guinea-pig outer hair cell membrane by activation of a Ca 2+-activated K − conductance.

Acetylcholine controls the gain of the voltage-to-movement converter in isolated outer hair cells.

Sensitivity of the electromotility of isolated guinea pig outer hair cells (OHCs) to acetylcholine (ACh) was examined. OHCs were held in a partitioning microchamber in a position so that their ciliary poles were inserted and their somatic length changes measured. Transcellular square-wave stimuli were delivered to record voltage-to-movement conversion of the cells. ACh was applied to the synaptic poles. The transfer function of electromotility intimated shifts of the membrane potential with concomitant gain changes: gain decreases when the membrane potential is shifted in hyperpolarization direction and gain increases when the membrane potential is shifted in the opposite direction due to application of ACh in 100-500 microM concentration. Clear gain increase of the electromotility in basal turn OHCs to ACh was observed, whereas inconsistent results for apical turn OHCs were found. The latter is probably due to the pharmacological dose of ACh used. This possibility is further supported by results in which ACh abolished the sensitivity of the magnitude response of electromotility to DC depolarizing bias.

Volume regulation in guinea pig outer hair cells and the role of intracellular calcium.

Single outer hair cells (OHCs) isolated from the guinea pig cochlea showed a regulatory volume decrease (RVD) after the initial cells swelling despite continued exposure to a hypotonic solution. The accompanying change of the intracellular Ca2+ concentration ([Ca2+]i) in the OHCs was investigated using the Ca(2+)-sensitive dye fura-2. Hyposmotic activation led to a [Ca2+]i increase which was accompanied by cell shortening and swelling. In a nominally Ca(2+)-free solution, however, [Ca2+]i was not significantly increased during hyposmotic activation although shortening and swelling of the OHCs were observed. These results suggest that the increase in [Ca2+]i during hyposmotic activation is mainly based on an influx of extracellular Ca2+.

C-type potassium channels in the lateral cell membrane of guinea-pig outer hair cells

The basolateral cell membrane of outer hair cells (OHC) from the mammalian cochlea is known to contain K +-channels. The prevailing type, a high-conductance K +-channel was further characterized in the present study in order to support its classification as C channel. OHC were isolated from the 3 rd and 4 th turn of the guinea-pig cochlea. Cell-attached and excised inside-out patches of the lateral cell membrane were investigated. The C-type channel had a selectivity for K + over Na + of 12:1 to 20:1 and displayed Goldman-type rectification and voltage-dependence of the open probability. The kinetics of both opening and closing could be described by time constants in the range of ms. The channel provides a calcium- and voltage-activated pathway through OHC lateral membranes for passive K + transport.

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.

Stretch-activated ion channels in guinea pig outer hair cells

Two types of stretch-activated (SA) ion channels have been found in the lateral wall of isolated outer hair cells (OHC) from the guinea pig cochlea. One type had a reversal potential of −12 mV and was non-selective to cations, passing Ca 2+ as well as monovalent ions. The channel had a conductance of 38–50 pS and the amplitude of the current through the open SA channel was independent of suction. The probability of the channel being open increased with applied suction and was voltage dependent with the maximum probability occurring at pipette potentials of −40 to −60 mV. The second type of SA channel had a conductance of approximately 150 pS and a reversal potential of approximately −50 mV. The ionic selectivity of this channel has not yet been determined, but it is probably K + selective. OHCs have been shown to undergo a slow change in length in response to acoustic stimulation directed at the lateral wall of the OHC. The SA channels reported here could affect the motile response by altering the membrane potential or by allowing the entry of free Ca 2+ which could lead to a change in OHC length through the interaction of actin and myosin. SA channels could also play an important role in regulating the osmotic pressure of OHC thereby influencing its electro-mechanical response.

Calcium channel in isolated outer hair cells of guinea pig cochlea

The physiological and pharmacological properties of the Ca 2+ channel in outer hair cells (OHCs) freshly isolated from guinea pig cochlea were investigated using a whole-cell patch-clamp technique. The Ca 2+ current ( I Ca) was activated from a membrane potential of −20 mV and reached peak value around +20 mV in external solution containing 20 mM Ca 2+ at a holding potential of −70 mV. The peak amplitude of I Ca increased in a hyperbolic manner with increasing extracellular Ca 2+ concentration. The ion selectively was Ba 2+ > > Ca 2+ ⩾Sr 2+. It was concluded that the Ca 2+ channel in OHCs of guinea pig is of the L-type.

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.

Serial Section Reconstruction of the Guinea Pig Outer Hair Cells as Studied with a High-Voltage Electron Microscope and a Computer-Graphic Display

The fine structural arrangement of the nerve endings on the guinea pig outer hair cells (OHCs) was studied by a serial sectioning technique by means of a high-voltage electron microscope and computer-graphic three-dimensional reconstruction. Sixty-nine series of consecutive sections were observed. Fifteen OHCs were located about 0.5 mm from the basal end (hook portion of basal turn) and 4 OHCs were seen at 6.0 mm, 20 OHCs at 8.0 mm (basal turn), 13 at 12.8 mm (second turn), 8 at 14.8 mm, 7 OHCs at 15.9 mm (third turn), and 2 OHCs at 17.5 mm (apical turn). Of 69 OHCs, 31 cells were from the first row, 22 from the second and 16 from the third row. The total number of nerve terminals on each OHC varied between 5 and 36. The OHCs with the least number of terminals were found in the hook portion, while the OHCs with the most numerous innervation were observed in the third row of the upper third turn. The afferent/efferent ratio (A/E ratio) of each OHC was evaluated and the OHCs were divided into two groups: Type A and Type B, depending upon the range of the A/E ratio. Thirty-five OHCs (50.7%) out of 69 were identified as being type A with an A/E ratio range of 0.5-1.5, while the remaining 34 OHCs (49.3%) were Type B cells with a greater A/E ratio than Type A cells. The detailed distribution of both types of cells was also studied along the entire length of the basilar membrane.