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Gross-potential recordings in mice lacking the Prestin gene indicate that compound action potential (CAP) thresholds are shifted by ∼45 dB at 5 kHz and by ∼60 dB at 33 kHz. However, in order to conclude that outer hair cell (OHC) electromotility is associated with the cochlear amplifier, frequency selectivity must be evaluated and the integrity of the OHC's forward transducer ascertained. The present report demonstrates no frequency selectivity in CAP tuning curves recorded in homozygotes. In addition, CAP input–output functions indicate that responses in knockout mice approach those in controls at high levels where the amplifier has little influence. Although the cochlear microphonic in knockout mice remains ∼12 dB below that in wild-type mice even at the highest levels, this deficit is thought to reflect hair cell losses in mice lacking prestin. A change in OHC forward transduction is not implied because knockout mice display non-linear responses similar to those in controls. For example, homozygotes exhibit a bipolar summating potential (SP) with positive responses at high frequencies; negative responses at low frequencies. Measurement of intermodulation distortion also shows that the cubic difference tone, 2f1–f2, is ∼20 dB down from the primaries in both homozygotes and their controls. Because OHCs are the sole generators of the negative SP and because 2f1–f2 is also thought to originate in OHC transduction, these data support the idea that forward transduction is not degraded in OHCs lacking prestin. Finally, application of AM1-43, which initially enters hair cells through their transducer channels, produces fluorescence in wild-type and knockout mice indicating transducer channel activity in both inner and outer hair cells.
In the mammalian cochlea, receptor cells of the organ of Corti are fully differentiated into two populations: inner (IHC) and outer hair cells (OHC). While IHCs serve as sensory receptors, sending information to the auditory nerve, OHCs have a motor function. Experimental evidence suggests that OHCs change their length in a voltage-dependent manner (Brownell et al. 1985; Kachar et al. 1986; Santos-Sacchi & Dilger, 1988). This electromotility is thought to provide a means for improving mammalian cochlear sensitivity and frequency selectivity (Ashmore, 1987; Dallos, 1992; Dallos & Fakler, 2002). Discovery of the OHC motor protein prestin, as well as the in vitro demonstration that prestin provides the molecular basis for electromotility (Zheng et al. 2000), both support the idea that prestin is associated with the cochlear amplifier (Davis, 1983). It has also been shown that Prestin gene and protein expression, as well as electromotility, exhibit the same developmental gradient (Zheng et al. 2000; Belyantseva et al. 2000). The importance of prestin for human hearing has also been reported by Liu et al. (2003) who demonstrated that a non-syndromic deafness is linked to a prestin mutation.
In spite of this evidence, an in vivo model is required in order to investigate the association between electromotility and the cochlear amplifier. This is because the OHCs are included in a tightly coupled feedback loop, which comprises the basilar membrane–OHC–tectorial membrane complex. Fortunately, development of the Prestin knockout mouse makes it possible to determine whether loss of OHC motor function affects cochlear sensitivity and/or frequency selectivity. Previous results (Liberman et al. 2002) indicate that OHCs isolated from F2 generation mice lacking prestin do not exhibit electromotility. There is also an ∼50 dB threshold shift for both auditory brainstem responses (ABRs) and otoacoustic emissions (OAEs). However, it is not known if frequency selectivity is also compromised. This information is required before one can conclude that prestin is associated with the cochlear amplifier.
It is also important to determine the functional status of mechanoelectrical transduction, which involves the mechanical gating of transducer channels located at the tips of the stereocilia (Hudspeth & Corey, 1977). In other words, an assessment of transducer function is required in order to learn if the loss of function phenotype reflects changes in forward, i.e. mechanoelectrical transduction, as opposed to the assumed reverse or electromechanical process. If prestin is a key component of the cochlear amplifier, then the functional changes observed in knockout mice should result from loss of OHC motor function, i.e. reverse or electromechanical transduction, and not from a change in forward transduction.
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CAP thresholds in knockout mice show a frequency-dependent, sloping loss between 35 and 60 dB. This loss of sensitivity in the CAP recorded at the round window is similar to that obtained by Liberman et al. (2002) in F2 generation mice using ABRs and OAEs. Although frequency selectivity was not measured in the Liberman et al. (2002) study, the loss of tuning in homozygotes is consistent with the idea that prestin is associated with the cochlear amplifier, i.e. those mechanisms that establish sensitivity and frequency selectivity in the peripheral auditory system. Our results in F4 generation mice also show essentially identical CAP responses in +/+ and +/− mice. This result is in conflict with the Liberman et al. (2002) study where a 6 dB threshold deficit was seen in heterozygotes. Taken together, the similarity of CAP thresholds and CAP tuning curves in F4 generation +/+ and +/− mice imply that one copy of the Prestin gene is sufficient for normal cochlear sensitivity and frequency selectivity.
In contrast to CAPs, which are summed neural responses, the CM reflects hair cell receptor currents. Although IHCs probably make some contribution to the CM, evidence suggests that this response is dominated by OHC receptor currents (Dallos & Wang, 1974; Dallos & Cheatham, 1976b; Patuzzi et al. 1989a). In addition, the CM recorded from the round window is a population response with contributing hair cell groups changing with stimulus frequency and level. It is therefore important to exercise caution when interpreting CM results. For example, CM data in normal animals suggests that low-level responses are more dependent on hair cell populations near the BF of the stimulus, whereas high-level responses are strongly dominated by cells near the round-window electrode even at low frequencies (Patuzzi et al. 1989a). This level of dependence is probably minimized in knockout mice where hair cell death begins at ∼3 weeks of age, and by 6 weeks of age IHC and OHC loss is observed over the basal 28% of the cochlea (Wu et al. 2004). To make matters worse, the reported profile of hair cell loss probably underestimates functional hair cell loss. Just because a cell is present does not mean that it is working properly (Steel & Bock, 1983).
Data in Fig. 4 indicate that CM responses in the present study for +/+ and +/− mice are commensurate with those obtained by Legan et al. (2000) who also used the same mixed 129SvEv/C57BL6 background to develop their α-tectorin knockout mice. Both studies show CM responses in wild-type controls that are much larger that those of Liberman et al. (2002). Independent of whether these differences are due to variations between F2 and F4 generations or whether the Liberman et al. (2002) data are abnormal, it is incorrect to assume that the similarity of CM responses in all three genotypes implies normal OHC transducer function (Liberman et al. 2002). In other words, one should not expect identical CM functions from ears that possess normal amplification and from those with impaired amplification. No matter how the amplification-providing feedback loop is opened, the system gain will be reduced and with it the input to the forward transducer. As a result, the transducer current, and thus the CM, will decrease. These reductions in the CM observed in F4 homozygotes could reflect a deficit in either forward or reverse transduction. Therefore, the status of the forward transducer was evaluated.
Results obtained in homozygotes using AM1-43 suggest that both inner and outer hair cells have open transducer channels. Although it is possible that this dye is taken up into hair cells via endocytosis (Nishikawa & Sasaki, 1996; Meyer et al. 2001; Griesinger et al. 2002), the time course is slower than that through functional transducer channels. While the latter occurs in seconds, endocytosis takes at least 2 min dye exposure in isolated sense organs (Si et al. 2003). In our case, however, the entire cochlea was incubated in AM1-43. While isolated organ segments were dissected for examination, this took place only after fixation in order to prevent damage to the transducer channels. Because of the longer diffusion time for the dye to enter scala media in intact preparations, one should probably not use estimates of AM1-43 entry via endocytosis that are derived from studies on organ segments. Furthermore, we have observed OHC fluorescence in knockout mice for cochlear incubation times as short as 68 s. Hence, the different time course, as well as the squelching of fluorescence in the presence of DHSM, suggests that the route of entry by AM1-43 for short exposures is via transducer channels. However, it is conceivable that transducer channels in homozygotes are fully open, allowing dye to enter, but not fully functional. If this were the case, one would not expect signal-dependent modulation of the channel. The presence of a CM argues against this possibility.
The physiological data also support the idea that forward transduction is normal in OHCs lacking prestin. CAP input–output functions at 6 kHz show near-normal magnitudes at high levels where minimal amplification is expected. However, the CM in the knockout mouse with the largest responses is still ∼12 dB down when compared to the wild-type mean. Reduction in the number of contributing hair cells in knockout mice could underlie this observation that homozygotes produce smaller maximal CM responses than do controls at high levels. In the presence of hair cell loss, the high-level CM in knockout mice will be diminished reflecting the reduction in the number of contributing hair cells, their increasing distance from the recording electrode and the complications associated with phase cancellation. For example, contributions from basal-turn OHCs in wild-type mice at 6 kHz are expected to grow linearly with level up to the point where the transducers of contributing hair cells begin to saturate. These contributions, obtained well below the BF of contributing OHCs, would not suffer reductions due to phase cancellation. As a result, the CM in wild-type mice at low frequencies is minimally influenced by the cochlear amplifier because the hair cells dominating the CM are responding linearly on the tails of their tuning curves. In contrast, the CM recorded from homozygotes is probably dominated by OHCs with BFs nearer to the stimulus frequency because of the incidental hair cell death occurring in these mice. As a result, increasing the level of a 6 kHz tone does not recruit as many OHCs from the base of the cochlea in knockout mice. Moreover, the summed responses from hair cells that do contribute are probably subject to phase cancellation. This argument suggests that the reduced CM is probably not due to a modification in forward transduction. This conclusion is supported by the existence of tip links between OHC stereocilia in knockout mice (Wu et al. 2004).
The presence of a bipolar SP is also consistent with normal OHC transducer function. Evidence from intracellular recordings suggests that OHCs respond to inputs below BF with negative, hyperpolarizing dc receptor potentials (Dallos et al. 1982; Russell & Sellick, 1983; Dallos, 1985, 1986; Cody & Russell, 1987), whereas IHCs never produce this polarity. These intracellular responses are reflected at the round window as negative SPs. It is also known that no negative SP is recorded in animals lacking OHCs in the basal half of the guinea pig cochlea due to kanamycin intoxication. Therefore, Dallos & Wang (1974) concluded that OHCs alone generate this SP polarity. This knowledge implies that SP responses, obtained at what are low frequencies in the mouse, should be asymmetrical in the negative direction when OHCs with viable transducers, basal to the BF location of the stimulus, contribute to the responses.
The presence of a cubic difference tone at ∼20 dB below the primaries in both wild-type and knockout mice is also consistent with normal OHC transducer function. Measurement of intermodulation distortion in cochlear mechanics (Robles et al. 1997) or in neural responses (Dallos et al. 1980) suggests that the most prominent distortion product, 2f1–f2, is probably generated by OHCs. Because reverse transduction cannot produce this component in mice lacking prestin, present data support the idea that the cubic difference tone is associated with OHC mechanoelectrical transduction (Patuzzi et al. 1989b; Jaramillo et al. 1993), i.e. cochlear distortion originates in hair cell non-linearities (Goldstein, 1967; Dallos et al. 1969). The fact that intermodulation distortion in both knockout and wild-type mice is ∼20 dB down from the primaries also indicates that shortening of OHCs does not alter ciliary mechanics or the residual mechanical response (Santos-Sacchi, 2003).
Although results shown here are consistent with the idea that prestin and OHC motility are required for normal cochlear function, one cannot conclude that prestin alone is the cochlear amplifier. While we show that OHC motility is necessary for amplification, it is yet to be determined if it is sufficient or whether its interaction with other processes is required. This caveat is needed because removal of prestin makes it difficult to determine whether other elements in the feedback path contribute to the sensitivity and frequency selectivity of the peripheral auditory system (Géléoc & Holt, 2003a; Fettiplace & Ricci, 2003). This difficulty is exacerbated by the hair cell loss in the Prestin-null mouse, which complicates comparisons with controls.