Neurobiology of cochlear inner and outer hair cells: intracellular recordings

Hearing Research, Elsevier

22 (1986) 185-198

185

Neurobiology of cochlear inner and outer hair cells: intracellular

recordings

Peter Dallos Auditory Physiology Laboratory

and Department

of Neurobiology

and Physiology, Northwestern

University, Evanston, IL 60201, U.S.A.

Recordings were made in the low-frequency region (third and fourth turns) of the guinea pig cochlea from both inner (IHC) and outer (OHC) hair cells. Certain electrical characteristics of these cells that have been described before (P. Dallos (1985) J. Neurosci. 5, 1591-1608) are reviewed. These are resting membrane potentials, response magnitude and saturation, and response phase. A comparison of sensitivity and best frequency is provided for IHCs and OHCs that are located in the same cochlear region. New data are presented for the level-dependence of response phase and for the properties of response asymmetry and the resulting dc component. The effects of intracellular polarizing current upon ac response magnitude are shown for both cell types.

Introduction In the past, understanding of the properties of the peripheral auditory system tended to rely on two types of electrical measure. Traditionally, gross cochlear potentials (Dallos, 1973a) and single fiber discharges from the auditory nerve (Evans, 1975; Kiang, 1984) provided the data base for descriptions of the system’s operation. With the accomplishment of intracellular recording from cochlear inner hair cells (IHC), a new dimension of inquiry was made available (Russell and Sellick, 1977). During the past eight years much has been learned about the properties of IHCs (Russell and Sellick, 1978, 1983; Sellick and Russell, 1980; Sellick et al., 1983; Brown et al., 1983; Brown and Nuttall, 1984; Nuttall, 1985; Goodman et al., 1982; Patuzzi and Sellick, 1983; Dallos, 1985a). Intracellular recording from outer hair cells (OHC) is much more difficult, and until recently (Dallos, 1985a), only fragmentary information became available based on this recording technique (Dallos and Flock, 1980; Tanaka et al., 1980; Dallos et al., 1982; Dallos and Santos-Sac&i, 1983; Russell and Sellick, 1983; Cody and Russell, 1985). With the exception of our laboratory, all workers use an open Scala tympani, basal turn approach in either guinea pig or gerbil (Russell and Sellick, 1978). With this method the recording electrode passes through the basilar membrane toward either IHC or OHC. Because of the recording location, 0378-5955/86/$03.50

0 1986 Elsevier Science Publishers

the characteristic or best frequencies (BF) of the encountered hair cells range upward from 15 kHz. As a consequence, the properties of the ac receptor potentials cannot be quantitatively evaluated near the cell’s BF. To explain, low-pass filtering, due to the cell’s membrane capacitance and the high-impedance recording electrode, introduces significant and very difficult-to-assess attenuation of the ac response component with increasing signal frequency. In our method, the organ of Corti is approached through a fenestra in the lateral wall of the cochlea (Dallos et al., 1982). The electrode does not penetrate either the basilar membrane or the reticular lamina, but dwells only in the supporting cell matrix of the organ of Corti. The method allows measurement from any turn of the cochlea. Thus far we have recorded from the fourth, third and second turns, possessing approximate best frequencies of 200, 800 and 2400 Hz respectively. This paper is confined to reporting data from the fourth and third cochlear turns of the guinea pig. Our experimental techniques are described in Dallos et al. (1982) and Dallos (1985a). We have already described several basic properties of IHCs and OHCs from the low-frequency region of the guinea pig cochlea (Dallos et al., 1982; Dallos, 1985a). Some of these are briefly mentioned for the sake of a self-contained presentation. The major emphasis is, however, on some hitherto unpublished results and on the discussion of a few unresolved issues. Throughout the paper

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it is endeavored to compare and contrast the electrical behavior of inner and outer hair cells.

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Results and Discussion Resting membrane potentials There is an emerging consensus that the membrane potentials of unstimulated IHCs and OHCs tend to be different (Russell and Sellick, 1978, 1983; Cody and Russell, 1985; Dallos et al., 1982; Dallos, 1985a). Two examples each of stable resting potentials for the two hair cell types are presented in Figs. 1 and 2. It is apparent from these examples that the OHC membrane potential is roughly double that of the IHC. Because time-dependent changes in the membrane potential frequently occur (Dallos, 1985b), it is probably best to assess this measure immediately after penetrating the cell. The upper quartile of such initial resting potentials was reported as -42 mV for IHCs and -65.5 mV for OHCs (Dallos, 1985a). In considering probable underlying causes for this difference, we worked with the approximate figures of - 40 and - 70 mV, respectively. It could be shown from a study of an electrical circuit analog of the hair cells and their organ of Corti surround, that membrane potentials depend on the ratios of basolateral and apical membrane resistances of these cells (Dallos, 1983). Based on hair

IHC J”I4.I

Fig. 1. Two examples for the time-history membrane potentials during recording.

of inner

hair cell

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cell geometry, these resistance ratios could be crudely approximated. Predicted membrane potentials closely matched the experimentally observed values, suggesting the possible validity of the argument. Simply stated, the membrane potential (I’,) is established between the limits set by the positive endocochlear potential (E,) and the negative electrochemical potential (E,) governed by the basolateral membrane. A voltage divider, formed by the basolateral (perilymphatic face) and apical (endolymphatic face) membrane resistances determines where the actual membrane potential is set between E, and E,. For IHCs this point is established closer to E,, whereas the OHC membrane potential lies closer to E,. The most plausible reason for the difference is that the large basolateral surface of OHCs possesses relatively low resistance, thus pulling V, toward E,. In contrast, the more symmetrical distribution of resistances of

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IHC membranes allows the influence of the positive E, to be felt, thus yielding an effective depolarization of the IHC. It was noted that in the steady-state, very high OHC membrane potentials can sometimes be seen. The argument was made that such high V, (80 - 90 mV) is abnormal (Dallos, 1985b). This matter is further examined in a later section of this paper. Response magnitude, saturation and sensitivity For most stimulus conditions a combination of ac and dc receptor potentials are produced by both types of hair cell. In contrast to basal turn IHCs, those cells in the cochlear apex generate larger ac components than dc. In this section the more prominent ac component of the response is presented. The intracellul~ ac response, just as its better known extracellular counterpart, the cochlear microphonic (CM) potential, possesses no true threshold. At low sound levels this response tends to increase in proportion to the input. Depending on the relationship between signal frequency and the BF of the cell, the linear rise is superseded by saturation at particular sound levels. Departure from linearity first occurs at BF and spreads asymmetrically to lower and higher frequencies with increasing sound intensity. Both IHC and OHC exhibit this type of behavior. In a good II-K saturation begins to occur at BF at levels as low as 30 dB (sound level reference throughout the paper is 20 PPa). In contrast, far from BF a linear rise in the response can prevail at even 70 dB. We frequently see a decreasing response with increasing input at very high levels. The frequency dependence of receptor potential saturation is apparently a function of the location of the hair cell along the cochlear spiral. To demonstrate this, in Fig. 3 we offer iso-input ac magnitude plots for IHCs located in the third and fourth turns of the cochlea. The plots obtained at the lowest sound level as a function of stimulus frequency characterize the BF of the cells: 230 Hz for the 4th turn IHC and 900 Hz for its 3rd turn counterpart. The third turn plots reflect the familiar saturation pattern of this type of frequency response function, well known from a variety of other measures of activity at the auditory periphery. Thus at the level of the single unit response in

the auditory nerve (Rose et al., 1967), in the CM potential (Dallos, 1973b) and in the displacement of the basilar membrane (Rhode, 1971), one notes pronounced saturation around the BF and much more linear behavior below the BF. The latter corresponds to the ‘tail’ segment of auditory nerve fiber tuning curves. An interesting reversal of this trend is seen in the lowest frequency region of the cochlea. It is known from single fiber iso-input patterns (Rose et al., 1967) that for BFs below approximately 500 Hz the tail of the functions is on the ~~-frequency side. In other words, with increasing level the response functions expand more toward the high frequencies than the lows, in contrast to the pattern seen for fibers of higher BF. This reversal is reflected in the intracellular ac response as well, as attested to by the high intensity behavior of the plots in Fig. 3. Inasmuch as the existence of the tail segment in frequency response functions appears to be a common property of all types of measures in the auditory periphery, it is likely that our observations of IHC (and OHC as well; Dallos, 1985a) behavior simply reflect the nonlinear properties of the mechanical traveling wave (i.e., a reversed asymmetry of the very low frequency mechanical disturbance). The availability of both IHC and OHC responses from the same region of the cochlea permits the examination of one of the most comI

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of ac response plots from one third-turn inner hair cell. Parameter is sound pressure

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monly held assumptions about cochlear physiology. It has been a general and often stated belief that OHCs are significantly more sensitive than IHCs (Btkesy, 1970; Keidel et al., 1983; Wever, 1949). Arguments to this effect were based in part on anatomical considerations. Thus outer hair cells being located on the central, and presumably more mobile, part of the basilar membrane, are thought to produce a larger output at the same input level. Physiological evidence for greater OHC sensitivity derives from hair cell damage experiments (e.g., Davis et al., 1958; Dallos, 1973a) and from the two-segment nature of compound action potential magnitude functions (e.g., Davis, 1961; Yoshie, 1968; Aran, 1969). Alternative explanations for these observations are available in Dallos (1983) for the former set of data and in ijzdamar and Dallos (1976) for the latter. When the sensitivities of OHC-IHC pairs, derived from the same organ of Corti are examined, an unexpected result obtains. Using the ac receptor potential at the BF as the comparison measure, it is found that inner hair cells are more sensitive. A comparison of six pairs of cells indicates that IHCs require a median of 12.4 dB less sound pressure to produce a criterion ac response than OHCs (Dallos, 1985a). In the low-level, quasi linear response region one can simply compare response magnitudes as indicated in Fig. 4. The iso-input ac plots for an IHC and an OHC from the same organ of Corti reveal that at BF the magnitude difference at the lowest sound level used is 12.8 dB. It is concluded, based on these recordings, that at least in terms of their ability to produce ac intracellular receptor potentials, IHCs are approximately four times as sensitive as OHCs. This result should not be interpreted to mean that IHCs control the extracellular ac potential (CM). In fact, the evidence against that is very strong on both experimental (e.g., Dallos and Cheatham, 1976) and theoretical grounds (Dallos, 1983). Likewise, due to the punctate nature of IHC afferent innervation contrasted with the convergent pattern of OHC afferents (Spoendlin, 1969) if the latter do respond to sound they may do so at levels similar to IHC fibers. A final comparison in this segment pertains to the BF assignable to IHCs and OHCs that are located in the same cochlear region. It is our

12.8 dB 4

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Fig. 4. Comparison of ac response magnitudes for an IHC and an OHC from the same organ of Corti. Parameter is sound pressure level in decibels. The difference in sensitivity between the two hair cells is assessed from their response magnitude differences (12.8 dB) at BF for the 0 dB SPL condition.

general observation that the two types of hair cell do not differ significantly in their best frequencies. As shown in Fig. 5, at the lowest level where complete plots can be obtained both ac and dc plots peak at similar frequencies. In this example

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Fig. 5. AC and dc response magnitude plots from one IHC and one OHC from the same organ of Corti at low sound pressure levels. The purpose of the plots is to demonstrate the similarity in BF of ac and dc responses and the similarity in BF between IHC and OHC.

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the IHC and OHC are from the same organ of Corti, but not obtained in the course of a single electrode penetration. In other words, there is a longitudinal separation between the two cells. The BF of the IHC, determined either from ac or de measures, is 900 Hz, that of the OHC is 800 Hz, representing a difference of 0.17 octave. The greatest discrepancy that we have seen between BFs of IHCs and OHCs in a given preparation is approximately 0.25 octave. The estimated distance along the guinea pig’s cochlea that corresponds to a frequency change of one octave is 2.4 mm (Dallos, 1973b; Wilson and Johnstone, 1972). Since the longitudinal extent of the greatest difference in electrode location within a given preparation is about 0.5 mm (constrained by the size of the fenestra made in the cochlear bone) the expected range of BFs is computed as 0.21 octave. The agreement is reasonable, and we may conclude that IHCs and OHCs that are located at the same distance along the cochlea in a given animal possess similar best frequencies.

membranes (Russell and Sellick, 1978). The cutoff frequency of this low-pass filter is determined by the specific resistance and capacitance of the cells’ membranes. Inasmuch as our experimental data suggest different cutoff frequencies for IHC and OHC (470 and 1250 Hz respectively; Dallos, 1984, 1985a), it was concluded that the membrane properties of the two types of receptor cell may be different. Specifically, the peculiarities of the OHC basolateral membrane (Smith, 1978) were implicated in the higher cutoff frequency exhibited by this cell type. In Fig. 6 a direct comparison is provided between IHC and OHC response phase in a situation

Response phase

After considering some aspects of ac response magnitude, we now turn to issues pertaining to response phase. We have reported some phase characteristics before; here a few features are reemphasized and a new aspect, the level-dependence of phase is introduced. Phase is measured from fast fourier transformation of averaged responses to tone burst stimuli. In the past we presented phase characteristics of both types of hair cell responses referenced to the phase of the ac potentials measured in the extracellular fluid space within the organ of Corti (Dallos, 1984, 1985a). It was pointed out that OHC and organ of Corti (OC) response phases were essentially indistinguishable, except at the highest frequencies where the intracellular response exhibited a phase lag. In contrast, the IHC phase led that of the OC response by approximately 90” at very low frequencies. With increasing frequency this lead diminished and converted into a phase lag. In comparing the intracellular and extracellular responses one needs to anticipate one simple difference. This is due to the intracellular ac response being affected by the low-pass filter formed by the inherent parallel resistance-capacitance of biological

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Fig. 6. Phase difference between ac responses from one IHC and one OHC from the same organ of Corti as a function of frequency. Phase differences are averaged for 15, 30, 50 and 70 dB SPL presentations. Insets demonstrate the response waveforms (at 50 dB SPL) from both IHC (top traces) and OHC (bottom traces) at 100 Hz and at 800 Hz. Time scale is expanded for the latter traces in order to allow clear comparisons between IHC and OHC responses.

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where both hair cells were in the same organ of Corti. In line with our previous discussions, it is apparent that the intracellular ac potential recorded from the IHC leads that obtained from the OHC by approximately 90” at the lowest frequency used, 100 Hz. The left-hand inset, showing the recorded waveforms at this frequency, clearly depicts this phase difference between the two sinusoidal responses. With increasing frequency the phase lead diminishes and ultimately converts into a phase lag. The frequency where the phase difference between the responses of the two receptor cells reaches its minimum is, at least in the third turn, in the vicinity of the cells’ BF. Waveforms included in the right-hand inset attest to this. In the fourth turn the phase lead is maintained beyond the BF of the cells. Two issues need to be discussed relating to the low-frequency and the hip-fr~uency discrepancies, respectively, between IHC and OHC phases. Sellick and Russell (1980) noted the phase lead of the first-turn IHC receptor potential vis-a-vis the gross CM and adopted our interpretation (Dallos et al., 1972a; Dallos, 1973b), that IHCs are velocity-sensitive. We have often argued, based on indirect evidence obtained from CM data in kanamytin-poisoned cochleas with selective OHC destruc&ion, that while OHCs respond in proportion to basilar membrane displacement (BCkCsy, 1953) the stimulus to IHCs, at least at low frequencies, is basilar membrane velocity. These arguments were advanced to explain the phase lead and slope increase by 6 dB/octave in CM magnitude functions that could have been produced only by remaining IHCs after OHC destruction (Dallos, 1973b). It was proposed that the morphological substrate for this difference is to be sought in the tenuous or nonexistent attachment of IHC cilia to tectorial membrane (Kimura, 1966; Lim, 1972) and the consequent stimulation of IHCs by fluid flow in the subtectorial space (Dallos et al., 1972a; Billone and Raynor, 1973). It appears today that this explanation of the experimental results is still the most parsimonious means of reconciling the data with anatomical observations. The high-frequency phase difference between IHC and OHC requires a more ad-hoc ‘explafiation’. If the only low-pass filter affecting the intracellular response was the membrane resistance-

capacitance network, then in spite of differences in cutoff frequency, the high-frequency phase difference between IHC and OHC should converge toward zero. A residual phase accumulation for IHC responses signifies an additional stage of filtering associated with this cell type. In order to match both amplitude and phase responses, it was necessary to assume the presence of a second-order filter element, an underdamped pole, having a resonant frequency at around 1200 Hz (Dallos, 1984). Russell and Sellick (1983) also noted the need for additional filtering in the IHC response but assumed that a simple additional pole was sufficient to account for their data. This additional filter element was considered by them to be due to either the characteristics of the microelectrode or some frequency-dependent biasing of ciliary position. We proposed that the postulated resonance at 1,200 Hz may be due either to tectorial membrane-ciliary mechanics or voltage-dependent conductances of the cell’s basolateral membrane (Dallos, 1984). Further studies are needed to resolve this matter. In certain nonlinear systems the response phase is dependent on the stimulus level. This sort of nonlinearity in the auditory periphery was first identified at the level of auditory nerve responses. Anderson et al. (1971) while measuring phaselocking of low-frequency spike discharges, found a consistent dependence of the response phase on both the stimulus frequency-fiber BF relationship and stimulus level. It was shown that for frequencies below the BF increasing intensity yielded increasing phase lag, while conversely, above the BF more phase lead corresponded to more intense sounds. A similar relationship was identified in basilar membrane motion by Rhode and Robles (1974) and Sellick et al. (1982). It is now demonstrated in intracellular responses. In Fig. 7 relative phase measures are shown for ac responses from one IHC and one OHC. Trends for phase changes as a function of stimulus level are clear. At the cell’s BP there does not appear to be a systematic shift in response phase. Below the BF at all frequencies the phase lag increases, while above the BF at all frequencies the phase lead increases as the signal strength grows. The highest measured level, 90 dB SPL, is not included in the figure because the presence of higher harmonics in

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quencies, where the response waveform could be evaluated, the magnitude of depolarizing half cycles exceeded those in the hyperpolarizing phase, thus yielding a positive dc component. With the attenuation of the ac response component by the cells’ membrane filter and also by the recording electrode, in the BF region of their cells the dc response hugely dominated the cell’s output. We confirmed the existence of a depolarizing dc response in low-frequency IHCs and also demonstrated its presence in OHCs (Dallos et al., 1982). A recent report by Cody and Russell (1985) suggests that basal turn OHCs do not produce a depolarizing dc potential, except at the highest sound levels. Clearly, a potential conflict exists between our findings and, thus, some further consideration of response asymmetry is warranted. In Fig. 8 the maximal receptor potential in both

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Fig. 7. Relative response phase for one IHC and one OHC from the same organ of Corti as a function of sound pressure level. Parameter indicates the stimulus frequency. Plots at BF are shown with heavy square symbols. Individual plots are arbitrarily displaced by 20” increments at the 15 dB SPL level for clarity of presentation.

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the response which are commensurate with the fundamental component (Dallos and Oesterle, 1985) distorts the measured phase. Within the depicted range, however, the trends are very clear. Moreover, essentially the same phase behavior characterizes both IHCs and OHCs. It is apparent that the phase-nonlinearity described by Anderson et al. (1971) is a property of the basilar membrane mechanics (Rhode, 1971; Rhode and Robles, 1974; Sellick et al., 1982). Response asymmetry;

the dc component

One of the most striking observations reported by Russell and Sellick in their early publications (1977, 1978) was the preponderance of a dc response in IHCs from the high-frequency region of the cochlea. These authors noted that at low fre-

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Fig. 8. Peak ac receptor potential as a function of peak sound level. Responses for one IHC and OHC from the same organ of Corti in the third cochlear turn are shown at a stimulus frequency approximately equal to BF. Corresponding waveforms are shown on the bottom of the figure.

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depolarizing and hyperpolarizing phases is plotted as a function of linear peak sound pressure. Data are shown for an IHC and an OHC from the same cochlea for a frequency in the BF region. The averaged response plots from which the data points are derived are given at the bottom of the figure. While the response magnitudes at a given sound level are different for IHC and OHC, the character of the plots is essentially identical for both hair cells. Moreover, these patterns closely resemble those shown by Russell and Sellick (1983) for mammalian IHCs, by Hudspeth and Corey (1977) for saccular hair cells and by Crawford and Fettiplace (1981) for hair cells in the terrapin cochlea. The curves show an extreme degree of saturation for the hyperpolarizing response, similar to that indicated by Hudspeth and Corey (1977) and Crawford and Fettiplace (1981) but more abrupt than that reported by Russell and Sellick (1983). The saturation in the depolarizing direction is similar in all studies. The critical point here is the lack of difference between II-K and OHC patterns. From these data, and others reported earlier (Dallos et al., 1982; Dallos, 1985a), it seems apparent that low-frequency OHCs do produce a depolarizing dc response in the vicinity of their BF, even at low and moderate sound levels. To further underscore this contention, in Fig. 9 a similar plot is presented for a fourth-turn OHC. Response magnitudes are plotted for a frequency near the cell’s BF. Waveforms of the source-data

Peak

Pressure

are also shown in the right side of the figure. The pattern depicted in Fig. 9 closely resembles the function obtained for the higher frequency OHC (Fig. 8), albeit the fourth-turn OHC produces greater ac responses than those seen in the third cochlear turn of the guinea pig. It is concluded that OHCs in the low-frequency region of the cochlea have a similar propensity for producing depolarizing dc responses as IHCs. We have reported that well below the BF of OHCs the response asymmetry reverses, resulting in a hype~ola~~ng dc component (Dallos et al., 1982). In their recent report Cody and Russell (1985) agree with us on this count. They noted that first-turn OHCs produce negative dc potentials at low frequencies even though these cells gave only small depolarizing responses (5-6 mV, about the same magnitude as their figures show for the hyperpolarizing component) at higher frequencies at very high sound intensities. In Figs. 10 and 11 response mag~tude patterns are demonstrated for third-turn OHCs at various sound levels at frequencies well below BF. The first of these figures uses the same format that was presented before, i.e., the results are shown in a linear coordinate system. When so examined, the resulting pattern is not conspicuously different from that seen at BF. In fact, if the data points for the lowest sound level are ignored, then the only

(PO)

Fig. 9. Peak ac response magnitude as a function of peak sound level for a fourth turn OHC. Stimulus frequency is near the BF of the cell. Corresponding waveforms are shown on the right side of the figure.

Fig. 10. Peak ac response magnitude as a function of peak sound level for a third turn OHC. Stimulus frequency (200 Hz) is well below the BF of the cell. Corresponding waveforms are shown in the tight side of the figure. Vertical calibration bar: 10 mV for top two traces, 1 mV for bottom trace. Note inversion of the response asymmetry between 40 and 60 dB SPL.

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Fig. 11. Peak ac response magnitude as a function of peak sound level for OHCs from three different preparations. Stimulus frequency (150 or 200 Hz) is well below the BF of the cells. Note that both abscissa and ordinate are logarithmic to better represent the low-level response asymmetry which is in the h~e~ola~~ng direction, as opposed to the depolarizing asymmetry at high levels.

difference is the milder degree of saturation in the h~e~ol~zing response phase. The overall asymmetry is the same as seen at BF: the depolarizing phase is more prominent. However, an examination of the lowest level data points or the corresponding waveform clearly reveals that here the hyperpolarizing phase dominates. To see this relationship better, in Fig. 11 similar data for three OHCs are plotted in a log-log coordinate system in order to emphasize the smaller values. Notice that for the two lowest sound levels plotted, the hyperpolarizing responses are larger. At the next level the two phases are approximately equal, indicating a symmetrical response, while at all higher levels the depolarizing half-cycles are larger. The important conclusion from these data, supporting our earlier statements (Dallos et al., 1982; Dallos, 1985a) is that the hyperpolarizing response is both frequency- and level-dependent. We observe these negative dc components only at low frequencies and at low sound levels. Even at low stimulus frequencies the dc response becomes positive if the sound intensity is sufficiently high. This observation is again in contrast with the results of Cody and Russell (1985) who describe

only negative low-frequency dc response in their OHCs at any level. It is worth pointing out that our results are in harmony with the literature on gross co&ear potentials. To explain, the summating potential (SP), the presumed gross potential counterpart of the intracellular dc response, exhibits an orderly negative-to-positive transition with increasing frequency at any recording location within the cochlea (Honrubia and Ward, 1969; Dallos et al., 1972b). In recording cochlear potentials from the fourth turn of the cochlea after destroying all IHCs in the vicinity while maintaining some retention of OHCs, we have shown that both SP components are dominated by potentials produced by OHCs (Dallos and Cheatham, 1976). At least in theory (Dallos, 1984), this could be extrapolated to all cochlear locations. If so, then the prominent SP” and SP- components seen in the basal turn should also reflect greater contributions from OHCs. (Note that the polarity designation of the gross response is opposite to that of the intracellular response, inasmuch as the former is commonly measured from Scala media or with differential electrodes in the perilymphatic scalae.) If the Cody and Russell (1985) results prove to be correct then it must be assumed that the dominance of gross cochlear potentials shifts from apex to base between OHCs and IHCs, or that at least the dominance shifts as concerns the dc response component. It was in fact assumed at one time (Davis et al., 1958) that OHCs produce the SP+ whereas IHCs generate the SP-. Cody and Russell’s ideas are in line with this notion. In order to reconcile the third- and first-turn OHC data, another assumption is also required. This would indicate that either cochlear mechanics or the electrophysiolo~c~ characteristics of OHCs change in a radical manner along the length of the cochlea. The means of generation of the dc receptor potential components have not been established with certainty. Clearly, any asymmetrical nonlinearity in the transduction chain could contribute to the response. Such rectification could arise in cochlear hydrodynamic processes (Tonndorf, 1970), asymmetrical ciliary stiffness (Strelioff and Flock, 1984), or in a variety of electrical processes within the hair cells themselves. The two-component SP response was successfully mod-

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eled by McMullen and Mountain (1985) who needed to incorporate two nonlinearities: a voltage-dependent stiffness of the cilia and a nonlinear transducer conductance. The latter element is responsible for the positive intracellular de response in the model. Thus, in order to reconcile the McMullen-Mountain formulation and the CodyRussell data, it is necessary to assume that highfrequency OHCs possess linear transducer channels. Alternatively, if the two polarity dc responses are a consequence of mechanical excitation of the hair cells below their BF versus near the BF, then a radical lon~tudinal change in the excitation pattern needs to be formulated to account for the results. It would be conceivable that there is a tradeoff between the outer hair cells’ possible dual roles as receptors and effecters. The electrical characteristics of apical OHCs are certainly not too dissimilar from those of IHCs, and tend to suggest a receptor cell function. In contrast, the first-turn OHCs of Cody and Russell are unlikely to operate as receptors but may well function as effecters in a cochlear feedback circuit. One can adduce numerous examples from the literature on auditory nerve discharges that suggest longitudinal gradation of response properties. The most obvious of these is the change in shape of the single fiber tuning curve with best frequency. Lowfrequency tuning curves are basically V-shaped, generally lacking a well-defined tail segment, or possessing one on the ~~-frequency side. In eontrast, high-fr~uency tuning curves comprise two distinct segments: tips and low-frequency tails (Kiang, 1984). There may be an advantage in endowing the high-frequency region of the cochlea with a highly nonlinear feedback system using the OHCs as mechanical effecters (Kemp, 1980; Neely and Rim, 1983; Davis, 1983) and leaving the lowfrequency region without this mechanism. As attractive as the above concept of ‘longitudinal gradation’ may appear in reconciling two disparate sets of data, it ought to be tempered by the recognition that the dc cochlear response is highly susceptible to experimental manipulations and injury (Davis et al., 1958; Butler and Honrubia, 1963; Misrahy et al, 1957; Durrant and Gans, 1975). Two effects need to be considered. First, it was shown that OHCs often increase their membrane potential after microelectrode penetration to

the abnormally high - 80 to - 100 mV level (Dallos, 1985b). Concomitant with the increase in V, is a decrease in the stimulus-evoked response and its linearization (reduction of the dc component}. Second, a displacement of the cochlear partition toward the Scala vestibuli results in the reversal in polarity or elimination of the gross SP- (e.g., Davis et al., 1958). Recently Cheatham (unpublished results, 1985) demonstrated that probable pressure build-up in the cochlea during perfusion experiments eliminated the SP+ recorded around the BF from the Scala tympani (this is the analog of the normal SP- but recorded with opposite polarity in Scala tympani instead of the usual Scala media or Scala vestibuli sites) without affecting either the CM or the low-frequency SP-. The resulting condition, essentially normal CM and elimination of the normally dominant SP polarity around BF, is not unlike those reported by Cody and Russell (1985). It may be surmised that as a recording electrode moves through the basilar membrane in the OHC region it exerts a mechanical bias on the basilar membrane toward the Scala vestibuli. Since the IHCs are situated above the osseous spiral lamina in the first turn, electrodes aimed at them would be less likely to displace the basilar membrane, and thus create a mechanical bias resulting in altered recording conditions, It is probably fair to say that without further experiments, either using the lateral approach in turn one or the through-the-basilar membrane approach in the apex, one cannot resolve the question of de response generation by OHCs throughout the cochlea. Effects of polarizing current Russell (1983) and Nuttall (1985) have addressed the topic of how direct current polarization affects the response properties of high-frequency IHCs. Here similar material is offered for lowfrequency IHCs and also for OHCs. DC polarizing currents are passed into the cell through the recording electrode which is incorporated in a bridge circuit to permit recording during current injection (Mentor N-950). It is our experience that it is very difficult to accurately assess changes in the cell’s membrane potential that are due to current injection when using the single electrode method. The reason for this is that the

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electrode resistance changes during current injection and this change is very different when the tip is within a cell from when it is in the extracellular fluid space. While the no-current electrode resistance can be balanced within the cell with relative ease, the current-related impedance changes are hard to account for. Checking a high-impedance electrode for linear response with its tip in extracellular space is insufficient in our experience because of the different behavior within the cell. It is conjectured that the extensive presence of large organic anions within the cytoplasm interferes with the tip characteristics of the electrode during the passage of current. Because of these uncertainties, we do not present I-V curves, or represent response changes as a function of membrane potential. Instead, the response change is simply reported as a function of polarizing current. In Fig. 12 a representative plot is shown for an IHC. The behavior represented in the plot is common for both third and fourth-turn cells. Here the ac response magnitude is shown for BF stimulation. The data are obtained with a wave analyzer using continuous tones at 40-50 dB sound level. Within the current range used, the pattern can be fit with straight lines. Interestingly, hyperpolarizing current is more effective in increasing the response than depolarizing current is in decreasing it. The approximate values are 66%/nA for hyperpolarizing currents and 33%/nA for depolarizing ones. This sort of behavior may also be surmised from Nuttall’s first-turn IHC material

r

Polorlzing

250

Lii 6

Current

(nA)

Fig. 12. Percent ac response as a function of intracellular dc polarizing current recorded from an IHC. Stimulus is at BF at a modest sound level.

Polarizing

Current

(nA)

Fig. 13. Percent ac response as a function of intracellular dc polarizing current recorded from an OHC. Stimulus is at BF at a modest sound level.

(Nuttall, 1985). Comparisons with Russell’s (1983) interesting data are difficult because he reported magnitude changes as a function of V,. In Fig. 13 the companion plot is shown for an OHC. The character of this current dependence is quite different from that seen for IHCs. Apparently the hyperpolarizing currents are the less effective ones in producing a response change, whereas depolarization is very capable of decreasing the ac receptor potential. Because of the instability of the recording situation with large depolarizing currents, we have not been able to reverse the phase of the response. Such a reversal is expected to occur when the membrane potential reaches the magnitude of the endocochlear potential (Russell, 1983). It is likely that the differing propensity of the two current polarities to affect IHC and OHC responses is tied to the differences in resting membrane potentials between these cells. Since the membrane potential of OHCs is normally close to the electrochemical potential (E,) of the cell’s basolateral membrane, further hyperpolarization by extrinsic current is more difficult than for the partially depolarized IHC. Finally, a polarization experiment was performed on an OHC whose membrane potential stabilized at a high, abnormal level. Some aspects of the electrical behavior of this type of cell have been mentioned above and details were provided in Dallos (1985b). The polarization current de-

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livered into this cell had essentially no effect on the response. It is not known if larger currents (up to 2 nA were used) could have repolarized the cell, thereby restoring its response characteristics. It is apparent that we need to add to the catalog of deficiencies exhibited by this type of OHC the inability to affect the response by modest polarizing currents. This is not unexpected since the cell behaves as if its basolateral membrane were electrically transparent. In other words, the cell’s input resistance is very low and responses measured within it are essentially the same as seen in the extracellular fluid (Dallos, 1985b).

Data presented in this work complement our previous description of the electrical behavior of low-frequency cochlear hair cells in the guinea pig (Dallos et al., 1982; Dallas, 1985a). Several issues, presented before, are newly emphasized. Thus consistent differences between IHC and OHC membrane potentials are considered and it is argued that these are likely to be the result of differing resistance ratios between endolymphatic and perilymphatic cell surfaces for the two cell types (Dallos, 1983). AC response magnitudes are compared for the two receptors, indicating that, contrary to common assumptions, OHCs are not more sensitive than IHCs. In fact, when cell pairs from the same cochlea are compared, IHCs appear to be approximately four times as sensitive as OHCs in their production of ac receptor potentials. We have also shown that cells located at approximately the same location in the cochlea have similar best frequencies, whether they are inner hair cells or outers. Several nonlinear features of the response iharacteristics are reviewed and placed in new light. Thus it is demonstrated that high-level saturation is BF-dependent in that while third-turn cells appear most linear for frequencies below BF, their fourth-turn counterparts show more extended linearity above BF. This behavior is reminiscent of that seen in auditory nerve discharges. To demonstrate saturation in more detail, several peak response versus peak sound pressure plots are presented. These highlight the similarity at BF of IHC and OHC characteristics seen in both turns three

and four. Particularly noteworthy are the hard saturation shown for hyperpolarizing response phase and the pronounced asymmetry in the receptor potential that yields a net positive (depolarizing) dc response. These plots are also useful to reemphasize one of the significant differences between low-frequency IHCs and OHCs. While the former produces only depolarizing responses. OHCs can generate hype~ola~~ng dc components at low frequencies and low sound levels. Particular attention is paid to the potential discrepancy between our data (Dallos et al., 1982), showing a pronounced depolarizing dc response around BF in OHCs, and those of Cody and Russell (1985), whose first-turn OHCs do not generate positive de responses at moderate intensities. Various alternatives are considered for bringing these results into harmony. The phase behavior of ac responses from the two types of receptor cells is studied. Direct comparison of the receptor potentials from IHCs and OHCs in the same cochlea shows a well-demonstrated phase lead of the IHC response at very low frequencies. It is also seen that the phase difference between responses from IHC and OHC disappears around the cells’ BF. Response phase is dependent on both stimulus level and the relationship between BF and stimulus frequency. A nonlinear phase pattern is seen for both cell types which is similar to that demonstrated for auditory nerve responses (Anderson et al., 1971). Finally, some data are shown on the effect of direct current polarization of hair cells upon the magnitude of the receptor potential. There appears to be some difference between IHC and OHC behavior. Hyperpolarizing currents are more effective in changing the ac response of IHCs than depolarizing current injection. This is similar to the findings of Nuttall (1985). The opposite asymmetry characterizes OHC receptor potential changes with current polarity.

This research was supported by NINCDS Grant NS-08635. M.A. Cheatham, E. Oesterle and J. Santos-Sacchi participated in some phases of the data collection. I thank Drs. M.A. Cheatham and J. Siegel for their comments on the manuscript.

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