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The influence of transient asphyxia on receptor potentials in inner hair cells of the guinea pig cochlea
Hearing Research, 11 (1983) 373-384 Elsevier
373
The influence of transient asphyxia on receptor potentials in inner hair cells of the guinea pig cochlea I.J. Russell and E.M. Cowley Ethology and Neurophysiology Group, School of Biological Sciences, University of Sussex, Brighton BNI 9QG, U.K. (Received
17 May 1983; accepted
9 June 1983)
Intracellular recordings were made from inner hair cells in the basal turn of the guinea pig cochlea during transient asphyxia. During these periods the endocochlear potential was reversibly reduced, the hair cell resting membrane potential was slightly hyperpolarized and the cochlear microphonic potential was decreased, all with similar time courses; The eighth nerve compound action potential and DC and AC components of the inner hair cell receptor potential were strongly attenuated and with similar slow time courses. The asymmetrical low frequency receptor potentials became symmetrical, and this change was attributed to alterations in the mechanical properties of the organ of Corti. The desensitization of the cochlea during transient asphyxia was associated with the loss of asymmetry of the inner hair cell receptor potential. Key words:
cochlear
inner hair cell; asphyxia;
receptor
potential;
cochlea.
Introduction The exquisite frequency selectivity and sensitivity of the mammalian cochlea is reversibly reduced during brief periods of asphyxia. Threshold frequency tuning curves of nerve fibres [7] and the isoresponse frequency tuning curves of inner hair cell receptor potentials [2] become broader, and larger sound pressure levels are required to reach the chosen criteria upon which these curves are based. The experiments described in this paper examine the effects of transient asphyxia on the amplitudes of receptor potentials to high frequency tones close to the characteristic frequencies (CF) of the hair cells and on the symmetry of the receptor potentials to tones at low frequencies. Some of the findings described in this paper appear in a preliminary report [9]. Methods The techniques used in these experiments have been described in detail elsewhere [ 11,131. Young guinea pigs 180-260 g in weight were anaesthetized with pentobarbital sodium, Droleptan and Operidine (Janssen) according to a regime devised by 0378-5955/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
374
Evans [5]. Heart rate was monitored continuously, and just before intracellular recordings were begun, about 1 h before the termination of the experiment, the animals were injected with the muscle relaxant Flaxedil (M & B) (0.5 mg/kg) and artificially respired with a gas mixture of 95% oxygen and 5% carbon dioxide supplied by a constant pressure respirometer. Intracellular recordings were made from IHCs in the basal, high frequency turn of the cochlea, and the basilar membrane in this region was exposed through an oval opening made in the boney wall of the Scala tympani. Careful orientation of the head and siting of the opening made it possible to see the basilar membrane without draining perilymph from the Scala tympani. Tone bursts of 60-80 ms duration were delivered to the ear through a closed sound system consisting of a Beyer DT48 dynamic earphone coupled through a 4 mm diameter polythene tube to a hollow ear bar [Ill. This was equipped with a calibrated probe microphone for measuring sound pressure at the tympanic membrane. The phase and amplitude characteristics of the sound system were calibrated according to a previous description [lO,l l] and sound pressure levels (SPL) were measured in dB relative to 2 X
[lo]. With optimum frequency compensation of the electrodes, the high frequency cut-off of the recording system varied between 1700 and 4300 Hz. The microelectrodes were advanced under direct visual control with light from a fibre-optic light guide towards the basilar membrane by means of a hydraulic microdrive. Inner hair cells were identified by their large, asymmetrical receptor potentials at frequencies below 70 Hz, small resting potentials and large DC receptor potentials in response to tones close to their CF. A total of 23 cells displaying these characteristics were studied in these experiments. The amplitude of the phasic signals recorded through the electrodes changed when the tip of the electrode penetrated the cell. They grew in size for responses at low frequencies and became smaller for responses to high frequency tones [ 111. The changes were associated with phase lags of at least
375
?r/2 radians. These observations are an indication that most of the phasic signal is recorded across the resistance of the electrode and not its distributed capacitance. When an inner hair cell had been impaled, and its responses characterised, the respirator was switched off until the heart rate dropped to 12 beats/min when it was switched on again. This criterion was chosen as a means of standardizing the experiments. It was found that within a single preparation EP fell to about the same level each time the heart rate reached the criterion and that this effect was fully reversible. In these experiments it was possible to make simultaneous measurements of the intracellular and extracellular potentials with the exception of the endocochlear potential. The effects of transient asphyxia on this potential were measured separately after recording from the hair cells by advancing the microelectrodes into the Scala media. Signals from the recording electrodes were stored on a 4-channel FM tape recorder (Racal Store 4). They were analyzed with the aid of a Plessey 1 l-03 microcomputer and phasic signals were analysed with a pair of Brookdeal 9503-K lock-in amplifiers and an Omniphase unit [lo].
Results The influence of transient asphyxia on extracellular and intracellular DCpotentials and voltage responses to I5 kHz tones Transient asphyxia causes a progressive decline in the positive endocochlear potential recorded from the Scala media and a simultaneous, slow hyperpolarization of the inner hair cell resting membrane potentials. These changes are illustrated in the records shown in Figs. 1 and 2. The respirator was switched off at time zero on the records, and only switched back on again when the bradycardia, resulting from the asphyxia had reached the criterion of 12 heart beats/s. The time to achieve this varied between about 70-190 s in different preparations. The resting membrane potential and EP maintained their normal values of - 30 to - 45 mV (mean 41.6 mV) and + 80 to +90 mV, respectively, until about 40 s before the heart rate criterion was reached and they continued to decline for 5-15 s after the respirator had been switched on. The EP was decreased from about + 85 to about + 25 mV in
z ," 2_ a E Q
Off
On
+I00 0 -100
m 0
'
I
I
a
a
'
I
J
L L 100
I
u I
C
I
8
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L 200
Seconds Fig. 1. The effect of transient asphyxia on the endocochlear potential (upper trace) and the membrane potential of an inner hair cell (lower trace). The respirator was switched off at time zero, and on again 135 s later.
376
Off 50 0 -50
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I.. 0
1
. .
.
I 100
1
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Fig. 2. The effect of transient asphyxia on the membrane potential (EM), DC component (DC) and AC component (AC) of the inner hair cell receptor potential. Endocochlear potential (EP), cochlear microphonic (CM), and gross compound action potential of the auditory nerve (AP) from a single preparation. The respirator was switched off at time zero on the record, and on again 183 s later.
the preparation illustrated in Fig. 1 and to about 50% of its normal value of about 80 mV in the preparation illustrated in Fig. 2. The effect on the membrane potential of hair cells was less dramatic and these were hyperpolarized by about 10% of the normal values. The membrane potential in the cell shown in Fig. 1 was changed from -45 to about -52 mV and that shown in Fig. 2 altered from -30 to - 34 mV. The recovery from this decline was quite rapid and accompanied the tachycardia associated with the return of normal respiration. The EP returned initially to a level slightly above normal e.g. 95 mV compared with 85 mV in Fig. 2. This then returned to normal over a period of about 1 min. The resting membrane potential (Figs. 1 and 2) shows a more pronounced, shorter lasting overshoot during the recovery phase. This appears to be an artifact associated with the surge of the respiratory gas mixture when the respirator is turned on or possibly with the sudden increase in blood pressure associated with the return of normal respiration. The traces in Fig. 2, with the exception of the endocochlear potential, were recorded simultaneously, and they represent the CM and N, response recorded from the Scala tympani, and the AC and DC components of the receptor potential recorded intracellularly from an inner hair cell in response to a 15 kHz tone at 50 dB SPL. This frequency was close to the characteristic frequency of the hair cells in the region of the basilar membrane from which the recordings were made. All of these potentials decreased in amplitude during the transient asphyxia, but considerable variation existed in the extent and time course of their decline. The CM declined to about 65% of its normal response, with a time course which paralleled that of the endocochlear potential. The time courses of the N, response, AC and DC components of the inner hair cell receptor potential were all similar to each other, and were more profoundly influenced by the asphyxia than CM. They began their decline
311
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50
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30
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dB SPL
dB SPL
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0
.
l
.
a
l
l 0
I
1
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I
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40
50
60
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dB SPL Fig. 3. A & B. Amplitude-stimulus intensity relationships for the AC and DC components, respectively, of the receptor potentials recorded intracellularly from a hair cell in response to 15 kHz tones during normal respiration (+) and transient asphyxia (A). C. The relationship between the ratio of the AC and DC components of the receptor potential and stimulus intensity under conditions of normal respiration (0) and transient asphyxia (A) for a 15 kHz tone.
378
earlier, about 70 ms before the heart rate slowed to 12 beats/s, in the example shown in Fig. 2, and reached their minimum levels about 5 s after this. These cochlear responses returned to their normal response amplitude after a further period of about 60 s. The decline in the N, response and the DC component was virtually complete and both fell to amplitudes of less than 5% of their normal responses; however, the AC component of the receptor potential fell only to about 70%. The influence of asphyxia on the AC and DC components of the receptor potential Asphyxia caused changes in the relationships between the stimulus intensity and the amplitudes of the AC and DC components of the receptor potentials in seven hair cells, for which these relationships were examined. Examples of these relationships for single hair cells in response to tones at 15 kHz are shown in Figs. 3A and B. The slopes of the curves below 45 dB SPL are between l-1.5, while above this level they tend to saturate and the slopes become shallower (DC, 0.7; AC, 0.8). During asphyxia saturation disappears and the slope of the DC amplitude-intensity relationship becomes steeper. The similarity between the amplitude-intensity relationships of the AC and DC components gives rise to the relative independence of their ratio with the stimulus intensity (Fig. 3C). Thus the rectifying property of the transduction process which is responsible for the DC component is relatively independent of the stimulus intensity during normal respiration. This relationship is apparently altered during asphyxia. At low stimulus intensities the DC component is absent or very small and, as a consequence, the AC/DC ratio is large (Fig. 3C). This is reduced as the stimulus intensity is increased, possibly because rectification becomes intensity dependent during asphyxia. It is apparent that the processes which bring about the changes in the DC standing potentials in the cochlea (EP and hair cell resting potential) and in the hair cell receptor potentials are associated with each other, but are not the same. In an attempt to examine the processes responsible for the decline in the IHC receptor potentials, the voltage responses of the inner hair cells to low frequency tones were examined. The influence of transient asphyxia on the voltage response to low frequency tones The intracellularly recorded voltage response from an inner hair cell to low frequency, sinusoidal tones is asymmetrical about the resting membrane potential, with the amplitude of the depolarizing phase exceeding the hyperpolarizing phase by a factor of 2-5 (Fig. 4). The DC potential arising from the asymmetry was calculated from the difference in the time integrals of the depolarizing and hyperpolarizing phases and is plotted against time for the voltage response to a succession of 200 Hz tones at 3.5 dB SPL in the upper trace of Fig. 5. The respirator was switched off at time zero on these three traces and the resultant asphyxia caused the amplitude of the DC and peak to peak voltage response (AC) to decline with a similar time course. These responses slowly recovered to their normal values after the respirator was switched on about 70 s after the beginning of the experiment. The
379
Fig. 4. The voltage response of an inner hair cell to a 340 Hz tone burst at 93 dB SPL, before (upper trace), during (middle trace) and after (lower trace) transient asphyxia when normal respiration had been restored. Horizontal scale: small division 1 ms, large division 10 ms; vertical scale 5 mV. Each trace is an average of 8 sweeps.
0
25
50
75
100
125
Seconds
Fig. 5. The effect of transient asphyxia on the DC component (upper trace) and the AC component (middle trace) of the voltage response of an inner hair cell to a 200 Hz tone at 73.5 dB SPL. The lower trace is the response of the CM recorded from the Scala tympani. The respirator was switched off at time zero, and on again 70 s later.
380
CM (bottom trace) had a different time course, and began changing after the intracellular potential, and recovered more rapidly. The asphyxia reduced the peak to peak amplitude of the intracellular response to about 60% of its normal value, while the DC response dropped to zero and became slightly negative. It appeared that asphyxia caused the voltage response from an inner hair cell to become more symmetrical. This can be seen in the waveforms of Fig. 4 which were sampled before the beginning of the experiment illustrated in Fig. 5, immediately before the respirator was turned back on, and again 150 s after, when recovery was complete. The changes in the transfer characteristics of inner hair cells were examined before and after asphyxia. Inner hair cells, and their afferent innervation are excited during the rarefaction phase of the sound pressure [12,14]. Transfer functions were obtained by plotting the sound pressure level measured at the tympanic membrane (horizontal axis) against the intracellularly recorded receptor potential (vertical axis) (Fig. 6). Before the Lissajou figures could be obtained, the phases of the acoustic and receptor potential waveforms were normalised so that there was coincidence between the zero crossing phases of the rarefaction phase of the acoustic waveform and the depolarizing phase of the receptor potential (measured with respect to the resting membrane potential). Examples of transfer functions are shown in Figs. 6A-C. It can be seen in these figures that during transient asphyxia the transfer functions become virtually symmetrical about the operating point compared with that obtained during normal respiration. In addition the saturation associated with the phase of hyperpolarization disappears. The effect of asphyxia on the responses of inner hair cells to basilar membrane
Inner
A
hair cells respond
to the velocity
of the basilar
membrane
motion
at very low
1-e
Fig. 6. The relationship between the amplitude of the receptor potential recorded from three inner hair cells, and the sound pressure level recorded at the tympanic membrane in response to a 180 Hz tone at 93 dB SPL (A), 96 dB SPL (B), 98 dB SPL (C) during normal respiration (thick trace) and transient asphyxia (thin trace). Each curve is based on 192 samples of single cycle receptor potential and sound pressure waveforms which were the average of 8 sweeps. The amplitude of the receptor potential (vertical axis) is with reference to the membrane potential, -45 mV (A), - 45 mV (B), -40 mV (C), and the sign of the horizontal axis refers to the rarefaction phase of the sound pressure (positive) and its compression (negative).
381
1QG 18d17G16015014013a120IlOtoo90
Frequency
Hz
Fig. 7. The relationship between the zero crossing phase of the receptor potential and sound pressure measured at the tympanic membrane measured during normal respiration (+) and transient asphyxia (A).
frequencies (below a few hundred Hz 1121). This observation is based on phase measurements between the receptor potential, sound pressure level, and on the rate of growth of the receptor potential with frequency. At frequencies of about 400 Hz and below the basilar membrane of the guinea pig cochlea responds to the velocity of the stapes [14]. At these frequencies the displacement of the basilar membrane phase leads that of the stapes and the sound pressure level measured in the auditory meatus by 90”, and grows in amplitude at a rate of 6 dls/octave. This behaviour is also reflected in the CM which is believed to originate in the outer hair cells 13,121 and indicates their sensitivity to basilar membrane displacement. At frequencies below about 100 Hz, inner hair cell receptor potentials phase lead the sound pressure level by about 180” and grow at a rate of about 12 dB/octave, indicating a response to basilar membrane velocity. This phase lead and growth rate fall rapidly with increasing frequency due to the low pass electrical filter characteristics of the hair cell membrane, and a progressive change in the response of the inner hair cells from velocity to displacement [ 10,121. Fig. 7 shows the relationship between the phase of the receptor potential and sound pressure level measured in the auditory meatus for a single inner hair cell. This supports the notion that inner hair cells respond to the velocity of the basilar membrane, and that this response remains unchanged during asphyxia. The low frequency phase characteristics were plotted without adjustment for the low pass electrical filter characteristics of the recording system, which in this
382
instance had a cut-off frequency of 1.7 kHz, or for the electrical time constant of the hair cell which was 0.85 ms. This was measured from the rise and decay times of the membrane potential response to a 0.5 nA current pulse which was passed through the intracellular recording electrode and remained unchanged during asphyxia. The relationship between the asymmetry of the receptor potential and the membrane potential of inner hair cells It might be considered that the change in symmetry of the receptor potential during asphyxia is related to the decrease in EP and hence the change in driving voltage which occurs across the apical membranes of the inner hair cells. However, it must be pointed out that there is no close correlation in the time courses of the DC component of the receptor potential and EP during asphyxia (Fig. 2). To test the hypothesis that the asymmetry of the receptor potential waveform is a voltage-dependent phenomenon, the ratio of the peak to peak voltage response (AC component) and the DC component was measured in response to 200 Hz tones when the hair cell was depolarized and hyperpolarized by the injection of current pulses through the recording electrode (Fig. 8). It can be seen from this figure that this ratio is independent of membrane potential over a wide range of values.
L
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T 0
0
0
2
1
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-1 60
t
-1 LO
1
-120
-100
-80
-60
- LO
- 20
mV Fig. 8. The relationship between the ratio of the depolarizing and hyperpolarizing phases (D/H) of the voltage response of an inner hair cell to low frequency tones, and the membrane potential. The numbers adjacent to the records are the amounts of current (in nA) which were injected to produce the observed membrane potential. Each trace is a single sweep.
383
Discussion The most striking change demonstrated by inner hair cells during transient asphyxia is in their receptor potentials to low frequency tones. In comparison with the phase of hyperpolarization, the depolarizing phase is greatly reduced, and this changes the form of the receptor potential from rectified to almost symmetrical about the resting membrane potential (Figs. 4 and 6). This change in symmetry reduces the DC component of the receptor potential close to zero (Fig. 4). The voltage responses of the inner hair cells to high frequency tones were similarly affected by transient asphyxia in that, for low intensity tones, the DC component was reduced by a greater extent than the AC component. The decline of the DC component of the receptor potential, during transient asphyxia is also closely associated, in time and magnitude, with the loss of the compound action potential of the auditory nerve in response to high frequency tones. This reinforces the concept that the asymmetrical transducer conductance of inner hair cells [9] is essential for generating the DC component of the inner hair cell receptor potential in response to high frequency tones. At these frequencies the DC component initiates excitation of the auditory nerve fibres, presumably by providing the control voltage for release of transmitter at the hair cell afferent synapse. Transient asphyxia also causes reductions in the endocochlear potential and CM, and hyperpolarizes the resting membrane potentials of inner hair cells with time courses which are very similar (Figs. 1 and 2). This confirms the observations of Brown et al. [2] and supports a previous proposal [ 1 l] that the resting membrane potentials of inner hair cells are. depolarized by the presence of the endocochlear potential possibly by the constant leakage of potassium ions across the apical membranes. However, in view of the relatively small change in the resting membrane potential, in relation to the large decline in the endocochlear potential, this leakage conductance is small compared with that of the lateral and basal membranes of the inner hair cell. The endocochlear and hair cell resting potentials are believed to provide the driving voltage for the flow of receptor current across the transducer membrane [4,8]. In a linear system, any change in this voltage should result in a proportional change in both phases of the receptor potential. In fact the membrane properties of inner hair cells appear to be remarkably linear [8], and changes in the driving voltage across the apical membrane do not appear to alter the symmetry of the receptor potentials, at least over a limited range (Fig. 8). Furthermore, the very slight change in resting potential of the IHCs during asphyxia makes it unlikely that the change in symmetry is due to a redistribution of conductance channels. Thus the symmetrization of the receptor potentials during asphyxia is not due to changes in the electrical properties of the inner hair cells, but to other factors. According to the classical theory of Davis [4], vibration of the basilar membrane results in a shear displacement between the tectorial and basilar membranes, and the stereocilia of the inner hair cells are free to respond to the viscous flow of fluid between these two laminae [ 11,131. This response of the IHCs to the velocity of the basilar membrane remains unchanged during asphyxia. The tips of the stereocilia of
outer hair cells are embedded in the tectorial membrane and their rigidity could significantly control the coupling between the tectorial membrane and inner hair cells. According to a recent proposal [ 11, if the stereocilia of the outer hair cells resist displacement, then the inner hair cells would also react against this displacement, conceivably producing their biassed responses. Under these circumstances, the responses of inner hair cells would be expected to be quite nonlinear and depend intrinsically on active processes to maintain the integrity of the stereocilia on the outer hair cells. It is suggested that these active processes are interrupted during transient asphyxia and that this reduces the rigidity of the stereocilia, or their attachment to the cuticular plate [6]. The consequence of this is to reduce the bias in the shearing motion, which is the mechanical input to the inner hair cells, thereby decreasing the sensitivity and nonlinearity of the inner hair cell responses.
Acknowledgements We thank Drs. J.F. Ashmore, A.R. Cody and D.A. Lowe for helpful discussion and comments on the manuscript. This work was supported by a grant from the M.R.C.
References 1 Ashmore, J.F. and Russell, I.J. (1983): Sensory and effector functions of vertebrate hair cells. J. Submicrosc. Cytol. 15, 163-166. 2 Brown, M.C., Nuttall, A.L., Masta, RI. and Lawrence, M. (1983): Cochlear inner hair cells: Effects of transient asphyxia on intracellular potentials. Hearing Res. 9, 13 1- 144. 3 Dallas, P. (1973): Cochlear potentials and cochlear mechanics. In: Basic Mechanisms in Hearing, pp. 335-372. Editor: A.R. Msller. Academic Press, New York. 4 Davis, H. (1965): A model for transducer action in the cochlea. Cold Spring Harbor Symp. Quant. Biol. 30, 181-190. 5 Evans, E.F. (1979): Neuroleptanaesthesia for guinea pigs. Arch. Otolaryngol. 105. 185-186. 6 Flock, A. and Murray, E. (1977): Studies on sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. 83, 85-91. 7 Robertson, D. and Manley, G.A. (1974): Manipulation of frequency analysis in the cochlear ganglion of the guinea pig. J. Comp. Physiol. 91, 363-375. 8 Russell, I.J. (1983): The origin of the receptor potential in inner hair cells of the mammalian cochlea. Evidence for Davis’ theory. Nature (London) 301, 334-336. 9 Russell, I.J. and Ashmore, J.F. (1983): Inner hair cell receptor potentials investigated during transient asphyxia: A model for hair cell coupling. In: Hearing - Physiological Basis and Psychophysics. pp. 10-16. Editors: R. Klinke and R. Hartman. Academic Press, New York. 10 Russell, I.J. and Sellick, P.M. (1978): Intracellular studies of hair cells in the mammalian cochlea. J. Physiol. (London) 284, 261-290. 11 Russell, I.J. and Sellick, P.M. (1983): Low frequency characteristics of intracellularly recorded receptor potentials in guinea pig co&ear hair cells. J. Physiol. (London) 338, 179-206. 12 Sellick, P.M., Patuzzi, R. and Johnstone, B.M. (1982): Measurement of basilar membrane motion in the guinea pig using the Mossbauer technique. J. Acoust. Sot. Am. 72, 131-141. 13 Sellick, P.M. and Russell, I.J. (1980): The responses of inner hair cells to basilar membrane velocity during low frequency auditory stimulation in the guinea pig cochlea. Hearing Res. 2, 4399445. 14 Wilson, J.P. and Johnstone, J.R. (1975): Basilar membrane and middle-ear vibration in guinea pigs measured by capacitive probe. J. Acoust. Sot. Am. 57, 705-723.
373
The influence of transient asphyxia on receptor potentials in inner hair cells of the guinea pig cochlea I.J. Russell and E.M. Cowley Ethology and Neurophysiology Group, School of Biological Sciences, University of Sussex, Brighton BNI 9QG, U.K. (Received
17 May 1983; accepted
9 June 1983)
Intracellular recordings were made from inner hair cells in the basal turn of the guinea pig cochlea during transient asphyxia. During these periods the endocochlear potential was reversibly reduced, the hair cell resting membrane potential was slightly hyperpolarized and the cochlear microphonic potential was decreased, all with similar time courses; The eighth nerve compound action potential and DC and AC components of the inner hair cell receptor potential were strongly attenuated and with similar slow time courses. The asymmetrical low frequency receptor potentials became symmetrical, and this change was attributed to alterations in the mechanical properties of the organ of Corti. The desensitization of the cochlea during transient asphyxia was associated with the loss of asymmetry of the inner hair cell receptor potential. Key words:
cochlear
inner hair cell; asphyxia;
receptor
potential;
cochlea.
Introduction The exquisite frequency selectivity and sensitivity of the mammalian cochlea is reversibly reduced during brief periods of asphyxia. Threshold frequency tuning curves of nerve fibres [7] and the isoresponse frequency tuning curves of inner hair cell receptor potentials [2] become broader, and larger sound pressure levels are required to reach the chosen criteria upon which these curves are based. The experiments described in this paper examine the effects of transient asphyxia on the amplitudes of receptor potentials to high frequency tones close to the characteristic frequencies (CF) of the hair cells and on the symmetry of the receptor potentials to tones at low frequencies. Some of the findings described in this paper appear in a preliminary report [9]. Methods The techniques used in these experiments have been described in detail elsewhere [ 11,131. Young guinea pigs 180-260 g in weight were anaesthetized with pentobarbital sodium, Droleptan and Operidine (Janssen) according to a regime devised by 0378-5955/83/$03.00
0 1983 Elsevier Science Publishers
B.V.
374
Evans [5]. Heart rate was monitored continuously, and just before intracellular recordings were begun, about 1 h before the termination of the experiment, the animals were injected with the muscle relaxant Flaxedil (M & B) (0.5 mg/kg) and artificially respired with a gas mixture of 95% oxygen and 5% carbon dioxide supplied by a constant pressure respirometer. Intracellular recordings were made from IHCs in the basal, high frequency turn of the cochlea, and the basilar membrane in this region was exposed through an oval opening made in the boney wall of the Scala tympani. Careful orientation of the head and siting of the opening made it possible to see the basilar membrane without draining perilymph from the Scala tympani. Tone bursts of 60-80 ms duration were delivered to the ear through a closed sound system consisting of a Beyer DT48 dynamic earphone coupled through a 4 mm diameter polythene tube to a hollow ear bar [Ill. This was equipped with a calibrated probe microphone for measuring sound pressure at the tympanic membrane. The phase and amplitude characteristics of the sound system were calibrated according to a previous description [lO,l l] and sound pressure levels (SPL) were measured in dB relative to 2 X
[lo]. With optimum frequency compensation of the electrodes, the high frequency cut-off of the recording system varied between 1700 and 4300 Hz. The microelectrodes were advanced under direct visual control with light from a fibre-optic light guide towards the basilar membrane by means of a hydraulic microdrive. Inner hair cells were identified by their large, asymmetrical receptor potentials at frequencies below 70 Hz, small resting potentials and large DC receptor potentials in response to tones close to their CF. A total of 23 cells displaying these characteristics were studied in these experiments. The amplitude of the phasic signals recorded through the electrodes changed when the tip of the electrode penetrated the cell. They grew in size for responses at low frequencies and became smaller for responses to high frequency tones [ 111. The changes were associated with phase lags of at least
375
?r/2 radians. These observations are an indication that most of the phasic signal is recorded across the resistance of the electrode and not its distributed capacitance. When an inner hair cell had been impaled, and its responses characterised, the respirator was switched off until the heart rate dropped to 12 beats/min when it was switched on again. This criterion was chosen as a means of standardizing the experiments. It was found that within a single preparation EP fell to about the same level each time the heart rate reached the criterion and that this effect was fully reversible. In these experiments it was possible to make simultaneous measurements of the intracellular and extracellular potentials with the exception of the endocochlear potential. The effects of transient asphyxia on this potential were measured separately after recording from the hair cells by advancing the microelectrodes into the Scala media. Signals from the recording electrodes were stored on a 4-channel FM tape recorder (Racal Store 4). They were analyzed with the aid of a Plessey 1 l-03 microcomputer and phasic signals were analysed with a pair of Brookdeal 9503-K lock-in amplifiers and an Omniphase unit [lo].
Results The influence of transient asphyxia on extracellular and intracellular DCpotentials and voltage responses to I5 kHz tones Transient asphyxia causes a progressive decline in the positive endocochlear potential recorded from the Scala media and a simultaneous, slow hyperpolarization of the inner hair cell resting membrane potentials. These changes are illustrated in the records shown in Figs. 1 and 2. The respirator was switched off at time zero on the records, and only switched back on again when the bradycardia, resulting from the asphyxia had reached the criterion of 12 heart beats/s. The time to achieve this varied between about 70-190 s in different preparations. The resting membrane potential and EP maintained their normal values of - 30 to - 45 mV (mean 41.6 mV) and + 80 to +90 mV, respectively, until about 40 s before the heart rate criterion was reached and they continued to decline for 5-15 s after the respirator had been switched on. The EP was decreased from about + 85 to about + 25 mV in
z ," 2_ a E Q
Off
On
+I00 0 -100
m 0
'
I
I
a
a
'
I
J
L L 100
I
u I
C
I
8
I
L 200
Seconds Fig. 1. The effect of transient asphyxia on the endocochlear potential (upper trace) and the membrane potential of an inner hair cell (lower trace). The respirator was switched off at time zero, and on again 135 s later.
376
Off 50 0 -50
On
MP
05
I.. 0
1
. .
.
I 100
1
200
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Fig. 2. The effect of transient asphyxia on the membrane potential (EM), DC component (DC) and AC component (AC) of the inner hair cell receptor potential. Endocochlear potential (EP), cochlear microphonic (CM), and gross compound action potential of the auditory nerve (AP) from a single preparation. The respirator was switched off at time zero on the record, and on again 183 s later.
the preparation illustrated in Fig. 1 and to about 50% of its normal value of about 80 mV in the preparation illustrated in Fig. 2. The effect on the membrane potential of hair cells was less dramatic and these were hyperpolarized by about 10% of the normal values. The membrane potential in the cell shown in Fig. 1 was changed from -45 to about -52 mV and that shown in Fig. 2 altered from -30 to - 34 mV. The recovery from this decline was quite rapid and accompanied the tachycardia associated with the return of normal respiration. The EP returned initially to a level slightly above normal e.g. 95 mV compared with 85 mV in Fig. 2. This then returned to normal over a period of about 1 min. The resting membrane potential (Figs. 1 and 2) shows a more pronounced, shorter lasting overshoot during the recovery phase. This appears to be an artifact associated with the surge of the respiratory gas mixture when the respirator is turned on or possibly with the sudden increase in blood pressure associated with the return of normal respiration. The traces in Fig. 2, with the exception of the endocochlear potential, were recorded simultaneously, and they represent the CM and N, response recorded from the Scala tympani, and the AC and DC components of the receptor potential recorded intracellularly from an inner hair cell in response to a 15 kHz tone at 50 dB SPL. This frequency was close to the characteristic frequency of the hair cells in the region of the basilar membrane from which the recordings were made. All of these potentials decreased in amplitude during the transient asphyxia, but considerable variation existed in the extent and time course of their decline. The CM declined to about 65% of its normal response, with a time course which paralleled that of the endocochlear potential. The time courses of the N, response, AC and DC components of the inner hair cell receptor potential were all similar to each other, and were more profoundly influenced by the asphyxia than CM. They began their decline
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earlier, about 70 ms before the heart rate slowed to 12 beats/s, in the example shown in Fig. 2, and reached their minimum levels about 5 s after this. These cochlear responses returned to their normal response amplitude after a further period of about 60 s. The decline in the N, response and the DC component was virtually complete and both fell to amplitudes of less than 5% of their normal responses; however, the AC component of the receptor potential fell only to about 70%. The influence of asphyxia on the AC and DC components of the receptor potential Asphyxia caused changes in the relationships between the stimulus intensity and the amplitudes of the AC and DC components of the receptor potentials in seven hair cells, for which these relationships were examined. Examples of these relationships for single hair cells in response to tones at 15 kHz are shown in Figs. 3A and B. The slopes of the curves below 45 dB SPL are between l-1.5, while above this level they tend to saturate and the slopes become shallower (DC, 0.7; AC, 0.8). During asphyxia saturation disappears and the slope of the DC amplitude-intensity relationship becomes steeper. The similarity between the amplitude-intensity relationships of the AC and DC components gives rise to the relative independence of their ratio with the stimulus intensity (Fig. 3C). Thus the rectifying property of the transduction process which is responsible for the DC component is relatively independent of the stimulus intensity during normal respiration. This relationship is apparently altered during asphyxia. At low stimulus intensities the DC component is absent or very small and, as a consequence, the AC/DC ratio is large (Fig. 3C). This is reduced as the stimulus intensity is increased, possibly because rectification becomes intensity dependent during asphyxia. It is apparent that the processes which bring about the changes in the DC standing potentials in the cochlea (EP and hair cell resting potential) and in the hair cell receptor potentials are associated with each other, but are not the same. In an attempt to examine the processes responsible for the decline in the IHC receptor potentials, the voltage responses of the inner hair cells to low frequency tones were examined. The influence of transient asphyxia on the voltage response to low frequency tones The intracellularly recorded voltage response from an inner hair cell to low frequency, sinusoidal tones is asymmetrical about the resting membrane potential, with the amplitude of the depolarizing phase exceeding the hyperpolarizing phase by a factor of 2-5 (Fig. 4). The DC potential arising from the asymmetry was calculated from the difference in the time integrals of the depolarizing and hyperpolarizing phases and is plotted against time for the voltage response to a succession of 200 Hz tones at 3.5 dB SPL in the upper trace of Fig. 5. The respirator was switched off at time zero on these three traces and the resultant asphyxia caused the amplitude of the DC and peak to peak voltage response (AC) to decline with a similar time course. These responses slowly recovered to their normal values after the respirator was switched on about 70 s after the beginning of the experiment. The
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Fig. 4. The voltage response of an inner hair cell to a 340 Hz tone burst at 93 dB SPL, before (upper trace), during (middle trace) and after (lower trace) transient asphyxia when normal respiration had been restored. Horizontal scale: small division 1 ms, large division 10 ms; vertical scale 5 mV. Each trace is an average of 8 sweeps.
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Fig. 5. The effect of transient asphyxia on the DC component (upper trace) and the AC component (middle trace) of the voltage response of an inner hair cell to a 200 Hz tone at 73.5 dB SPL. The lower trace is the response of the CM recorded from the Scala tympani. The respirator was switched off at time zero, and on again 70 s later.
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CM (bottom trace) had a different time course, and began changing after the intracellular potential, and recovered more rapidly. The asphyxia reduced the peak to peak amplitude of the intracellular response to about 60% of its normal value, while the DC response dropped to zero and became slightly negative. It appeared that asphyxia caused the voltage response from an inner hair cell to become more symmetrical. This can be seen in the waveforms of Fig. 4 which were sampled before the beginning of the experiment illustrated in Fig. 5, immediately before the respirator was turned back on, and again 150 s after, when recovery was complete. The changes in the transfer characteristics of inner hair cells were examined before and after asphyxia. Inner hair cells, and their afferent innervation are excited during the rarefaction phase of the sound pressure [12,14]. Transfer functions were obtained by plotting the sound pressure level measured at the tympanic membrane (horizontal axis) against the intracellularly recorded receptor potential (vertical axis) (Fig. 6). Before the Lissajou figures could be obtained, the phases of the acoustic and receptor potential waveforms were normalised so that there was coincidence between the zero crossing phases of the rarefaction phase of the acoustic waveform and the depolarizing phase of the receptor potential (measured with respect to the resting membrane potential). Examples of transfer functions are shown in Figs. 6A-C. It can be seen in these figures that during transient asphyxia the transfer functions become virtually symmetrical about the operating point compared with that obtained during normal respiration. In addition the saturation associated with the phase of hyperpolarization disappears. The effect of asphyxia on the responses of inner hair cells to basilar membrane
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Fig. 6. The relationship between the amplitude of the receptor potential recorded from three inner hair cells, and the sound pressure level recorded at the tympanic membrane in response to a 180 Hz tone at 93 dB SPL (A), 96 dB SPL (B), 98 dB SPL (C) during normal respiration (thick trace) and transient asphyxia (thin trace). Each curve is based on 192 samples of single cycle receptor potential and sound pressure waveforms which were the average of 8 sweeps. The amplitude of the receptor potential (vertical axis) is with reference to the membrane potential, -45 mV (A), - 45 mV (B), -40 mV (C), and the sign of the horizontal axis refers to the rarefaction phase of the sound pressure (positive) and its compression (negative).
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Fig. 7. The relationship between the zero crossing phase of the receptor potential and sound pressure measured at the tympanic membrane measured during normal respiration (+) and transient asphyxia (A).
frequencies (below a few hundred Hz 1121). This observation is based on phase measurements between the receptor potential, sound pressure level, and on the rate of growth of the receptor potential with frequency. At frequencies of about 400 Hz and below the basilar membrane of the guinea pig cochlea responds to the velocity of the stapes [14]. At these frequencies the displacement of the basilar membrane phase leads that of the stapes and the sound pressure level measured in the auditory meatus by 90”, and grows in amplitude at a rate of 6 dls/octave. This behaviour is also reflected in the CM which is believed to originate in the outer hair cells 13,121 and indicates their sensitivity to basilar membrane displacement. At frequencies below about 100 Hz, inner hair cell receptor potentials phase lead the sound pressure level by about 180” and grow at a rate of about 12 dB/octave, indicating a response to basilar membrane velocity. This phase lead and growth rate fall rapidly with increasing frequency due to the low pass electrical filter characteristics of the hair cell membrane, and a progressive change in the response of the inner hair cells from velocity to displacement [ 10,121. Fig. 7 shows the relationship between the phase of the receptor potential and sound pressure level measured in the auditory meatus for a single inner hair cell. This supports the notion that inner hair cells respond to the velocity of the basilar membrane, and that this response remains unchanged during asphyxia. The low frequency phase characteristics were plotted without adjustment for the low pass electrical filter characteristics of the recording system, which in this
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instance had a cut-off frequency of 1.7 kHz, or for the electrical time constant of the hair cell which was 0.85 ms. This was measured from the rise and decay times of the membrane potential response to a 0.5 nA current pulse which was passed through the intracellular recording electrode and remained unchanged during asphyxia. The relationship between the asymmetry of the receptor potential and the membrane potential of inner hair cells It might be considered that the change in symmetry of the receptor potential during asphyxia is related to the decrease in EP and hence the change in driving voltage which occurs across the apical membranes of the inner hair cells. However, it must be pointed out that there is no close correlation in the time courses of the DC component of the receptor potential and EP during asphyxia (Fig. 2). To test the hypothesis that the asymmetry of the receptor potential waveform is a voltage-dependent phenomenon, the ratio of the peak to peak voltage response (AC component) and the DC component was measured in response to 200 Hz tones when the hair cell was depolarized and hyperpolarized by the injection of current pulses through the recording electrode (Fig. 8). It can be seen from this figure that this ratio is independent of membrane potential over a wide range of values.
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Discussion The most striking change demonstrated by inner hair cells during transient asphyxia is in their receptor potentials to low frequency tones. In comparison with the phase of hyperpolarization, the depolarizing phase is greatly reduced, and this changes the form of the receptor potential from rectified to almost symmetrical about the resting membrane potential (Figs. 4 and 6). This change in symmetry reduces the DC component of the receptor potential close to zero (Fig. 4). The voltage responses of the inner hair cells to high frequency tones were similarly affected by transient asphyxia in that, for low intensity tones, the DC component was reduced by a greater extent than the AC component. The decline of the DC component of the receptor potential, during transient asphyxia is also closely associated, in time and magnitude, with the loss of the compound action potential of the auditory nerve in response to high frequency tones. This reinforces the concept that the asymmetrical transducer conductance of inner hair cells [9] is essential for generating the DC component of the inner hair cell receptor potential in response to high frequency tones. At these frequencies the DC component initiates excitation of the auditory nerve fibres, presumably by providing the control voltage for release of transmitter at the hair cell afferent synapse. Transient asphyxia also causes reductions in the endocochlear potential and CM, and hyperpolarizes the resting membrane potentials of inner hair cells with time courses which are very similar (Figs. 1 and 2). This confirms the observations of Brown et al. [2] and supports a previous proposal [ 1 l] that the resting membrane potentials of inner hair cells are. depolarized by the presence of the endocochlear potential possibly by the constant leakage of potassium ions across the apical membranes. However, in view of the relatively small change in the resting membrane potential, in relation to the large decline in the endocochlear potential, this leakage conductance is small compared with that of the lateral and basal membranes of the inner hair cell. The endocochlear and hair cell resting potentials are believed to provide the driving voltage for the flow of receptor current across the transducer membrane [4,8]. In a linear system, any change in this voltage should result in a proportional change in both phases of the receptor potential. In fact the membrane properties of inner hair cells appear to be remarkably linear [8], and changes in the driving voltage across the apical membrane do not appear to alter the symmetry of the receptor potentials, at least over a limited range (Fig. 8). Furthermore, the very slight change in resting potential of the IHCs during asphyxia makes it unlikely that the change in symmetry is due to a redistribution of conductance channels. Thus the symmetrization of the receptor potentials during asphyxia is not due to changes in the electrical properties of the inner hair cells, but to other factors. According to the classical theory of Davis [4], vibration of the basilar membrane results in a shear displacement between the tectorial and basilar membranes, and the stereocilia of the inner hair cells are free to respond to the viscous flow of fluid between these two laminae [ 11,131. This response of the IHCs to the velocity of the basilar membrane remains unchanged during asphyxia. The tips of the stereocilia of
outer hair cells are embedded in the tectorial membrane and their rigidity could significantly control the coupling between the tectorial membrane and inner hair cells. According to a recent proposal [ 11, if the stereocilia of the outer hair cells resist displacement, then the inner hair cells would also react against this displacement, conceivably producing their biassed responses. Under these circumstances, the responses of inner hair cells would be expected to be quite nonlinear and depend intrinsically on active processes to maintain the integrity of the stereocilia on the outer hair cells. It is suggested that these active processes are interrupted during transient asphyxia and that this reduces the rigidity of the stereocilia, or their attachment to the cuticular plate [6]. The consequence of this is to reduce the bias in the shearing motion, which is the mechanical input to the inner hair cells, thereby decreasing the sensitivity and nonlinearity of the inner hair cell responses.
Acknowledgements We thank Drs. J.F. Ashmore, A.R. Cody and D.A. Lowe for helpful discussion and comments on the manuscript. This work was supported by a grant from the M.R.C.
References 1 Ashmore, J.F. and Russell, I.J. (1983): Sensory and effector functions of vertebrate hair cells. J. Submicrosc. Cytol. 15, 163-166. 2 Brown, M.C., Nuttall, A.L., Masta, RI. and Lawrence, M. (1983): Cochlear inner hair cells: Effects of transient asphyxia on intracellular potentials. Hearing Res. 9, 13 1- 144. 3 Dallas, P. (1973): Cochlear potentials and cochlear mechanics. In: Basic Mechanisms in Hearing, pp. 335-372. Editor: A.R. Msller. Academic Press, New York. 4 Davis, H. (1965): A model for transducer action in the cochlea. Cold Spring Harbor Symp. Quant. Biol. 30, 181-190. 5 Evans, E.F. (1979): Neuroleptanaesthesia for guinea pigs. Arch. Otolaryngol. 105. 185-186. 6 Flock, A. and Murray, E. (1977): Studies on sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. 83, 85-91. 7 Robertson, D. and Manley, G.A. (1974): Manipulation of frequency analysis in the cochlear ganglion of the guinea pig. J. Comp. Physiol. 91, 363-375. 8 Russell, I.J. (1983): The origin of the receptor potential in inner hair cells of the mammalian cochlea. Evidence for Davis’ theory. Nature (London) 301, 334-336. 9 Russell, I.J. and Ashmore, J.F. (1983): Inner hair cell receptor potentials investigated during transient asphyxia: A model for hair cell coupling. In: Hearing - Physiological Basis and Psychophysics. pp. 10-16. Editors: R. Klinke and R. Hartman. Academic Press, New York. 10 Russell, I.J. and Sellick, P.M. (1978): Intracellular studies of hair cells in the mammalian cochlea. J. Physiol. (London) 284, 261-290. 11 Russell, I.J. and Sellick, P.M. (1983): Low frequency characteristics of intracellularly recorded receptor potentials in guinea pig co&ear hair cells. J. Physiol. (London) 338, 179-206. 12 Sellick, P.M., Patuzzi, R. and Johnstone, B.M. (1982): Measurement of basilar membrane motion in the guinea pig using the Mossbauer technique. J. Acoust. Sot. Am. 72, 131-141. 13 Sellick, P.M. and Russell, I.J. (1980): The responses of inner hair cells to basilar membrane velocity during low frequency auditory stimulation in the guinea pig cochlea. Hearing Res. 2, 4399445. 14 Wilson, J.P. and Johnstone, J.R. (1975): Basilar membrane and middle-ear vibration in guinea pigs measured by capacitive probe. J. Acoust. Sot. Am. 57, 705-723.