Apical Outer Hair Cells Research Articles

TMC2 Modifies Permeation Properties of the Mechanoelectrical Transducer Channel in Early Postnatal Mouse Cochlear Outer Hair Cells.

The ability of cochlear hair cells to convert sound into receptor potentials relies on the mechanoelectrical transducer (MET) channels present in their stereociliary bundles. There is strong evidence implying that transmembrane channel-like protein (TMC) 1 contributes to the pore-forming subunit of the mature MET channel, yet its expression is delayed (~>P5 in apical outer hair cells, OHCs) compared to the onset of mechanotransduction (~P1). Instead, the temporal expression of TMC2 coincides with this onset, indicating that it could be part of the immature MET channel. We investigated MET channel properties from OHCs of homo- and heterozygous Tmc2 knockout mice. In the presence of TMC2, the MET channel blocker dihydrostreptomycin (DHS) had a lower affinity for the channel, when the aminoglycoside was applied extracellularly or intracellularly, with the latter effect being more pronounced. In Tmc2 knockout mice OHCs were protected from aminoglycoside ototoxicity during the first postnatal week, most likely due to their small MET current and the lower saturation level for aminoglycoside entry into the individual MET channels. DHS entry through the MET channels of Tmc2 knockout OHCs was lower during the first than in the second postnatal week, suggestive of a developmental change in the channel pore properties independent of TMC2. However, the ability of TMC2 to modify the MET channel properties strongly suggests it contributes to the pore-forming subunit of the neonatal channel. Nevertheless, we found that TMC2, different from TMC1, is not necessary for OHC development. While TMC2 is required for mechanotransduction in mature vestibular hair cells, its expression in the immature cochlea may be an evolutionary remnant.

Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals

The base of the cochlea is more vulnerable to trauma than the apex as seen in the pattern of hair cell damage by cisplatin or aminoglycosides. The differential vulnerability is maintained in organotypic cultures exposed directly to these drugs, suggesting there may be an intrinsic difference in sensitivity to damage along the cochlear spiral. We therefore investigated the survival capacity of isolated outer hair cells and strips dissected from different turns of the guinea pig organ of Corti in short-term culture. Cells were stained with fluorescent indicators of viable or dead cells, calcein-AM and ethidium homodimer. After 5 h at room temperature, up to 90% of outer hair cells from the apex survived, but less than 30% from the base. In contrast, basal inner hair cells remained viable, and supporting cells survived for at least 20 h. The difference in survival capacity between basal and apical outer hair cells coincided with a significantly lower level of the antioxidant glutathione in basal outer hair cells compared with apical outer hair cells. This suggested that basal outer hair cells may be more vulnerable to free-radical damage than apical outer hair cells. The survival of basal outer hair cells was significantly improved by addition of the radical scavengers n-acetyl cysteine, p-phenylenediamine, glutathione, mannitol or salicylate. The protection by antioxidants implies that the accelerated death of basal outer hair cells is due to free-radical damage. The results support an intrinsic susceptibility to free radicals that differs among cochlear cell populations. This differential provides a rational explanation for base-to-apex gradients observed in various forms of cochlear pathology.

Effects of gentamicin and pH on [Ca 2+] i in apical and basal outer hair cells from guinea pigs

Aminoglycosides are widely used antibiotics and frequently produce acute ototoxicity. In this study we attempted to comparatively investigate the effects of gentamicin on Ca 2+ influx of apical and basal outer hair cells (OHCs) isolated from guinea-pig cochlea. Since the solution of gentamicin sulfate salt is acidic (pH 3.1–3.3), we also explored the effect of external acidification on Ca 2+ influx. By means of fura-2 microspectrofluorimetry, we measured the intracellular calcium concentration ([Ca 2+] i) of OHCs bathed in Hanks’ balanced salt solution (pH 7.40) during either a resting state or high K +-induced depolarization. Our results show that at the resting state, the baseline [Ca 2+] i in apical OHCs (94±2.0 nM) was slightly lower than that in basal OHCs (101.1±2.4 nM). By contrast, the increase in [Ca 2+] i evoked by high K + depolarization in apical OHCs was about two-fold greater than that in basal OHCs. Nifedipine (30 μM) abolished the increased [Ca 2+] i in both types of OHCs, suggesting that Ca 2+ influx was mainly through L-type Ca 2+ channels of OHCs. While gentamicin and extracellular acidification (pH 7.14) can separately attenuate this increase in [Ca 2+] i in both types of OHCs, their suppressive effects are additive in basal OHCs, but not in apical OHCs. The implications of these findings are that: (1) apical and basal OHCs behave differently in response to depolarization-increased [Ca 2+] i, and (2) basal OHCs are more vulnerable to the impairment of Ca 2+ entry during depolarization by a combination of gentamicin and extracellular acidification, which is correlated with the clinical observation that ototoxicity of aminoglycosides at the basal coil of OHCs is more severe than that at the apical coils. Moreover, the possibility that extracellular acidification may enhance the acute ototoxic effects of aminoglycosides should be considered especially in topical applications.

Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat.

1. The non-linear capacitance (Cnon-lin) of postnatal outer hair cells (OHCs) of the rat was measured by a patch-clamp lock-in technique. Cnon-lin is thought to result from a membrane protein that provides the molecular basis for the unique electromotility of OHCs by undergoing conformational changes in response to changes in membrane potential (Vm). Protein conformation is coupled to Vm by a charged voltage sensor, which imposes Cnon-lin on the OHC. Cnon-lin was investigated in order to characterize the surface expression and voltage dependence of this motor protein during postnatal development. 2. On the day of birth (P0), Cnon-lin was not detected in OHCs of the basal turn of the cochlea, whilst it was 89 fF in apical OHCs. Cnon-lin increased gradually during postnatal development and reached 2.3 pF (basal turn, P9) and 7.5 pF (apical turn, P14) at the oldest developmental stages covered by our measurements. The density of the protein in the plasma membrane, deduced from non-linear charge movement per membrane area, increased steeply between P6 and P11 and reached steady state (4200 e- microm-2) at about P12. 3. Voltage at peak capacitance (V) shifted with development from hyperpolarized potentials shortly after birth (-88.3 mV, P2) to the depolarized potential characteristic of mature OHCs (-40.8 mV, P14). This developmental difference in V was also observed in outside-out patches immediately after patch excision. During subsequent wash-out V shifted towards the depolarized value found in the adult state, suggesting a direct modulation of the molecular motor. 4. Thus, the density of the motor protein in the plasma membrane and also its voltage dependence change concomitantly in the postnatal period and reach adult characteristics right at the onset of hearing.

Apical hair cells and hearing

Hair cells located in the cochlear apex were destroyed by low-frequency noise exposure or by application of a − 140 °C cryoprobe to the bony wall of the apical turn. Complementary lesions that spared apical hair cells were produced by treatment with ototoxic antibiotics. Behavioral thresholds were measured in monkeys, chinchillas, and guinea pigs before and after these treatments. The results showed (1) with loss of up to 80% of apical outer hair cells, thresholds were unchanged; (2) with complete loss of both outer and inner apical hair cells, thresholds shifted approximately 20 dB at low frequencies; and (3) with complete loss of hair cells except in the apex, some hearing remained, with better hearing at low frequencies. Addition of a high-pass masker always elevated high-frequency thresholds, but only elevated low-frequency thresholds when all apical hair cells had been destroyed, indicating that basal hair cells can respond to low-frequency stimuli. Compared to data from more basal portions of the cochlea, these data suggest that functional differences exist between the base and apex, with comparable damage in the apex resulting in less hearing impairment. [Work supported by NINCDS grants NS05785 and NS25564.]

Selective destruction of the outer hair cells in the chinchilla.

In the study of cochlear function an animal model in which apical outer hair cells could be destroyed with retention of inner hair cells would be valuable. Our object was to attempt to reproduce a reported method in a scientific manner. Behavioural response thresholds to pure tones were recorded in 7 monaural chinchillas. In 5, Kanamycin was administered in ototoxic dosage until the thresholds deteriorated. The other 2 served as controls. Cochlear histology was assessed from embedded surface preparations. In only one of the 5 animals was the aim achieved. In this animal outer hair cells were absent virtually throughout the cochlea and inner hair cell retention was basal. Ths thresholds were elevated on average 40 dB throughout the frequency range. This is in keeping with current concepts of cochlear function.