Post-stimulatory effects of direct current stimulation of the cochlea on auditory nerve activity

Hearing Research, Elsevier

HRR

21

36 (1988) 21-40

01122

Post-stimulatory

effects of direct current stimulation on auditory nerve activity

Hugo Cousillas

1*2,

Robert

B. Patuzzi ’ and Brian M. Johnstone

I Department of Ph.vsiology, Uniuersi& of Western Australia, Nedlands, Australia. and ’ INSERM de ~Audition C.H. R. St. Char/es. Montpellier, France (Received

of the cochlea

21 December

1987; accepted

- 254, Lahoratoire

1 de Neurohiologie

12 June 1988)

Glass micro-electrode recordings from the spiral ganglion of the basal turn of the guinea pig cochlea have been obtained before, during and after negative (cathodic) current injection into Scala tympani. Electrical stimulation with currents between 100 pA and 900 QA produced a marked increase in firing rate of the afferent neurons for the first 3 min of electrical stimulation. This was followed by a fall in firing rate to rates near or below the prestimulato~ spontaneous rate if stimulation continued. Continuous electrical stimulation lasting 5 or 10 min reduced neural sensitivity to acoustic stimulation. Although threshold elevation was greatest for sound frequencies near the characteristic frequency of each neuron, thresholds could also be elevated at lower frequencies on the tail of the frequency-threshold tuning curve. After electrical stimulation a fall in the amplitude of the low-frequency microphonic recorded at the round window was also observed, indicating a disruption of the outer hair cell transduction. These effects were highly localized in the basal turn near the site of current injection, and were not associated with any significant structural changes in the organ of Corti. except after stimulation with very high current intensities. Neuron

single, Electrical

stimulation;

Co&ear

nerve;

Tuning

Introduction Previous work has shown that injection of direct current into the cochlea alters the firing of auditory neurons, either modulating the neural firing rate during the application of current (Konishi et al., 1970; Teas et al., 1970) or, if the current intensity is sufficiently high, producing a reduction in the spontaneous firing of the nerves after current ceased (Schreiner et al., 1986). It has been suggested that such electrical stimulation may form the basis of a treatment of tinnitus in human subjects (Cazals et al., 1978; Portmann et al., 1979; Aran, 1981; Aran and Cazds, 1981). The mechanisms producing these changes in neural firing are not well understood, although it has been suggested that the post-stimulatory effects of

Correspondence 10: Dr Robert B. Patuzzi, Department of Physiology, University of Western Australia, Nedlands, W.A. 6009, Australia. 037%5955/88/$03.50

0 1988 Elsevier Science

Publishers

electrical stimulation may involve the exhaustion of neurotrans~tter from the inner hair cells (Schreiner et al.? 1986). If this suggestion is correct, electrical stimulation may be useful in the study of the neurochemistry of synaptic transmission in the cochlea. The purpose of these experiments was, therefore, to determine whether the changes in single neuron activity produced during and after current stimulation were consistent with an exhaustion of the afferent transmitter. In particular. we would expect exhaustion of neurotransmitter from the inner hair cells to produce threshold elevation for all stimulus frequencies used to evoke neural firing, similar to the changes seen after disruption of neurotransmission by Mg*+ perfusion into the cochlea (Siegel and Relkin, 1984). To test this hypothesis we have measured electrical activity from single primary afferent neurons in the first turn of the guinea pig spiral ganglion before, during and after cathodic electrical stimulation of the Scala tympani. The neural responses to acous-

B.V. (Biomedical

Division)

22

tic stimuli were characterised by frequency threshold curves (FTC), rate-level functions and per&stimulus time histograms. Our results indicate that the electrical stimulation we have used had a very localized but complicated effect on the cochlear response to acoustic stimulation. It did not produce a simple elevation of neural threshold across the full frequency range of the single neuron FTC, as would be expected from simple transmitter exhaustion, but produced exaggerated threshold elevation near the characteristic frequency (CF) of each neuron. It affected the function of outer hair cells (OHCs), the afferent and efferent neurons of the organ of Corti, and possibly inner hair cells (IHCs). Microscopic examination also indicated that there was a very small range of current intensity over which reversible changes to the cochlea could be produced. High levels of current produced structural damage to the sensory epithelium. This suggests that the clinical use of such current injection for tin&us suppression may have severe practical limitations. M&erials and method

Physiology

Eighty young, pigmented guinea-pigs (200-300 g) were used in these experiments. Following premedication with atropine sulphate (0.65 mg/kg)

animals were anaesthetized with intraperitoneal injections of Nembutal (25 mg/kg sodium pentobarbitone) followed by intramuscular injection of Innovar-Vet (0.2 mg/kg Fentanyl; Droperidol, 10 mg/kg). The animals were tracheotemized and artificially ventilated with a mixture of air (50%) and carbogen (50%). They were then paralysed with intramuscular injections of -4lloferin (alcuro~um chloride, 2.5 mg/kg). The rectal temperature was maintained at 37 o C using an electric heating blanket. Supplementary doses of analgesic and anaesthetic were given at 90 min intervals. A surgical approach similar to that described by Robertson and Manley (1974) was used (Fig. 1). To determine the compound cochlear action potential (CAP) threshold a wire electrode was positioned on the bone near the round window. Extracellular recordings of single neuron activity within the spiral ganglion of the first turn were obtained using glass micropipettes filled with the dye ‘Chicago Sky Blue’ to enhance visibility. Their impedances ranged from 100 to 200 MS2. The acoustic stimulus was a 100 ms tone burst with a 0.5 ms rise/fall time. The sound source was an half-inch condenser microphone (Brtiel and Kajer 4134) driven in reverse. Single neuron FTC% ratelevel functions and per-i-stimulus time histograms were determined automatically using a computer. Single neuron threshold was defined as an increase of 20 spikes/s in neural firing rate above its spontaneous activity. MICROELECTRODE SPIRAL

\ RE

STIMULATINQ

ELECTRODE

CALA

NlAL HOLE TYMPANI

GAVGLION

TYMPANl

GANGLION

IN SCALA WALL

CALA

MEDIA

Fig. 1. Schematic representation of the spirai ganglion approach and the positioning of the different electrodes. Adapted from Robertson and Johnstone (1979).

23

The negative or cathodic direct current stimulus was injected through a glass micropipette with a broken tip (diameter 50 pm) filled with a saline solution (0.9% NaCl). This stimulus electrode was positioned near the basilar membrane in Scala tympani in the region of the recording area. If a cochlear opening had been made for single neuron recordings the stimulus electrode was introduced through this opening, otherwise it was introduced through a small hole in the round window. The current return electrode was positioned subcutaneously on the abdomen of the guinea pig. A Grass stimulator (S44) was used as a current source, and the delivered current was monitored and adjusted throughout all current injection procedures. Current intensities were between 100 PA and 900 p A, and of either 5 or 10 min duration in the studies of the gross cochlear responses, or 10 min only for studies of single neuron activity. Histologv After each experiment, both cochleae were fixed with glutaraldehyde (3%) in cacodylate buffer (0.1 M) and left overnight in the fixative solution. The cochleae were then washed in cacodylate buffer (2 x 15 mm), followed by a postfixation in 1% osmium tetroxide (1 h) and two further washings (15 min each) in the buffer. For transmission electron microscopy (TEM) dehydration in ethanol and propyleneoxide was followed by embedding in araldite (3 days at 60” C). Ultrathin sections were made using an ultratome (LKB Ultratome III). These sections were stained (uranyl acetate and lead citrate) and then observed in a TEM (Phillips 300) at 80 kV tension. For scanning electron microscopy (SEM) dissection was completed in 70% alcohol. Scala vestibuli was opened and Reissner’s membrane and the tectorial membrane were removed. Ethanol dehydration was followed by final dehydration in a Polaron critical point dryer. The cochleae were then metal coated and observed in a SEM (Phillips 505). Results

Effects on gross cochlear potentials CAP visual detection thresholds for acoustic stimuli over the range 2-30 kHz (Fig. 2) were

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Fig 2. Typical CAP threshold before and after electrical stimulation (300 PA) through the round window (RW). and in scala tympani (ST) through the cochlear opening for the spiral ganglion recordings. Data are from two different preparations.

recorded before and after the negative (cathodic) polarization of Scala tympani. Current intensity in these initial experiments was fixed at 300 PA and was either delivered through a small hole in the round window, or through the cochlear opening through the Scala tympani wall for the spiral ganglion approach. For both stimulation sites CAP thresholds, determined within the 3 min immediately after electrical stimulation, were elevated relative to pre-stimulus thresholds for sound frequencies above 10 kHz (Fig. 2). The maximum threshold elevation produced seemed not to depend greatly on the duration of the current stimulus. Both the 5 min and 10 min electrical stimula-

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Fig. 3. Typical recovery following 5 and 10 min window. The results are current

curves for CAP threshold at 1X kHz current stimulation through the round from two different preparations for a intensity of 300 PA.

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tion could produce maximum threshold elevations of about 45 to 50 dB (Fig. 3). The most apparent difference between the two durations was in the recovery time following stimulation. After 5 min of stimulation the recovery of CAP threshold occurred in 2 stages. The first stage of the recovery was fast, with the hearing loss recovering by about 25 dB during the first 3 min. The second stage was slower, lasting about 15 min (Fig. 3). After 10 min of stimulation, the recovery occurred in only one slow stage lasting about 40 min (Fig. 3). For the subsequent study of single neuron responses an electrical stimulus lasting 10 min was used because the prolonged recovery time allowed collection of more data during the recovery period. After electrical stimulation the amplitude of the round window microphonic was also reduced. A 200 Hz pure tone acoustic stimulus was used to monitor the condition of the OHCs of the first turn because this avoided the complications associated with stimuli of higher frequency, such as phase cancellation (Whitfield and Ross, 1965; Patuzzi, 1987). Although 200 Hz does not correspond to the normal range of CF in the basal turn, there is evidence that the 200 Hz round window microphonic at high intensities is produced by the OHCs of this region (for example, Cheatham and Dallos, 1982). Following a 5 min electrical stimulus (300 PA) delivered through the round window, the intensity function for the 200 Hz microphonic

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Fig. 4. The effect of 5 min of current stimulation (300 PA) through the round window on the input-output function for the round window microphonic evoked by a 200 Hz pure tone. The microphonic amplitude fell by an approximately equal percentage across the intensity range used immediately after current stimulation (0 min). and recovered to levels near the pre-stimulatory values in 10 min.

measured at the round window fell almost uniformly by 50 to 70% across the intensity range used (Fig. 4). This fall in microphonic was associated with the elevation of CAP threshold described previously. The microphonic recovered rapidly following current stimulation, almost to its level prior to stimulation. No detailed study of the correlation between the reduction in the microphonic amplitude and the elevation of the CAP thresholds has been performed. Following a 10 min electrical stimulation the microphonic was reduced by about the same amount as for the 5 min stimulation, but recovered more slowly. Effects on single neuron activity Effect on single neuron action potentials The amplitude of the single neuron action potentials recorded from the spiral ganglion decreased monotonically with the intensity of the current stimulus and, if the current intensity was sufficiently high, the action potentials were suppressed entirely. The current level necessary to produce a 50% reduction in amplitude varied between preparations, and ranged from 200 I_LA to 400 PA. This variation in the efficacy of the electrical stimulus was also evident in the changes following electrical stimulation, as will be discussed in more detail later. We found this reduction in amplitude of the single neuron action potential to be a more reliable indicator of the probable disruption after electrical stimulation than the current intensity per se. Although we have not investigated the reasons for this variation in detail, it was presumably due to a variation in the portion of delivered current that actually passed through the organ of Corti. For example, the effect of current of a particular intensity injected through the round window was greater than for the same current delivered through the cochlear opening. In this case it is likely that the seepage of perilymph from the cochlear opening created an additional current return pathway on the external cochlear wall which shunted current from the organ of Corti. Effects on single neuron firing rate The firing rate of afferent neurons could be measured during electrical stimulation if the cur-

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Fig. 5. Four examples of the changes in neural firing rate during electrical stimulation. The presentation of the electrical stimutus is indicated by the horizontal bar. The firing rate of neurons increased markedly for the first 2 to 3 min of stimulation, but fell to levels near or below the pre-stimulatory spontaneous rate for the remainder of the stimulus presentation.

rent intensity was insufficient to abolish the action potential. In such cases the firing rate increased transiently to rates between 2 and 5 times the pre-stimulus spontaneous firing rate (Fig. 5). Within 2 to 3 min of the start of electrical stimulation, however, the firing rate fell to levels near or below the initial spontaneous rate, and normally remained at or near this reduced level for the duration of the electrical stimulus. On cessation of the stimulus spontaneous activity could return almost immediately (Fig. 5). The post-stimulatory spontaneous rate could be above or below the pre-stimulatory spontaneous rate. Although it was clear that the spontaneous activity was not dramatically reduced by electrical stimulation that did not abolish the action potentials, we were unable to hold sufficient neurons during the 10 min of electrical stimulation to quantify the effect of electrical stimulation on

spontaneous firing rate. Only one data point could be obtained from each animal. Higher current intensities that abolished the action potentials had a more dramatic influence on spontaneous firing rate, producing a silent period immediately after the current was discontinued during which no spontaneous firing was evident. This silent period ranged from 30 s to 6 m. Again, because we could not be sure that the spontaneous firing after this silent period was from the same neuron, and because we could only obtain one data point from a single animal, we have been unable to quantify the effect of the higher current intensities on spontaneous firing rate. Effects on single neuron tuning The results presented in this report between 15 kHz and 20 kHz. Following

had CFs electrical

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Fig. 6. Frequency threshold curves obtained from 9 preparations before (0) and after (x) 10 min of electrical stimulation at the current intensities indicated. Data obtained from different neurons before and after electrical stimulation are indicated (0). Note that threshold elevation following electrical stimulation is greater near CF than on the low frequency tail of the tuning curves. Note also the variation in the threshold elevation between preparations stimulated with equal current intensities. Where high frequency data is not available a broken line of nominal slope 400 dB/octave has been added to aid comparison.

stimulation of sufficient intensity the neural sensitivity to acoustic stimuli was reduced. As with the effect of electrical stimulation on the action potential amplitude, the amount of threshold elevation varied between preparations for the same current intensity. Fig. 6 presents tuning curves obtained before and after electrical stimulation in 9 typical animals for various current intensities, and illustrates the variation in the effect of the electrical stimulus for equal intensities of the delivered current. In some animals contact with a particular neuron was lost during the 10 min of electrical stimulation. Although the neural activity recorded subsequently may have been from the same neuron there was no way to be sure. In such cases we have compared responses from the cell encountered at the commencement of electrical stimulation with

those of the cell first encountered following cessation of the electrical stimulus. This possible change in neuron is unlikely to account for much of the differences observed in the neural tuning before and after electrical stimulation, since the tuning curves observed normally within a single hole in the spiral ganglion vary little (see Fig. 7 and Robertson, 1983, 1984). Although elevation of neural threshold could be produced at all frequencies across the FTC. the greatest elevations were observed for frequencies near the CF of each neuron (Fig. 6). This can be seen if threshold at the tip of the tuning curve before and after electrical stimulation is plotted against the threshold elevation on the tail. This has been done in Fig. 8, in which tip threshold was calculated as the arithmetic mean of threshold sound pressure at CF (before electrical stimula-

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Fig. 7. Frequency threshold curves obtained from 3 neurons recorded successively from the same hole in the spiral ganglion. Note the small variation in threshold on the tail of the FTC which is typical of recordings from the spiral ganglion.

tion) and 1 kHz either side of CF. The elevation of the FTC tail was calculated by taking the arithmetic mean of threshold elevation at frequencies from 1 kHz to 10 kHz, in 1 kHz increments. Each line segment of Fig. 8 represents results from one animal, with the end points of each segment defined by the tip threshold and zero tail elevation before electrical stimulation, and the new tip

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Fig. 8. Neural threshold at the tip of the frequency threshold curve before and after electrical stimulation plotted against average change on the low-frequency tail of the threshold curve. Data are from 22 preparations. Tip threshold is defined as the arithmetic mean of threshold at CF and 1 kHz above and below CF. Average tail elevation is the arithmetic mean of threshold elevations between 1 kHz and 10 kHz in 1 kHz increments. Note that changes in threshold near CF were greater than those on the tail, as indicated by line segment slopes less than 0.5 dB/dB.

threshold and average tail elevation after presentation of the electrical stimulus. It can be seen from Fig. 8 that the changes at the tip are generally greater than those on the tail, as indicated by line segment slopes much less than the 1 dB/dB slope expected if the changes were the same. Similar exaggerated changes at the tip of the FTC have been observed after Furosemide intoxication (Sewel. 1984). It has been suggested that this effect can be explained by the presence of a frequency-invariant desensitization of IHCs and/ or afferent neurons, and the frequency-dependent. nonlinear growth of the transverse vibration of the organ of Corti (Sellick et al., 1982). For example. if a larger vibration of the organ of Corti were required to reach neural threshold after trauma. the compressive growth of vibration only found near CF would require a greater increase in sound intensity to reach neural threshold than would be the case on the tail. This could potentially produce a broadening of the FTC. Such a mechanism is obviously unable to explain the elevation of the tip of the FTC in the absence of threshold elevation on the tail (for example Fig. 6. units HO08 and H016). Even in cases where tail elevation was observed, the change on the tail was generally too small to explain the threshold elevation at CF (for example, units PC057 and HO02 of Fig. 6). as will be discussed in more detail later. If these arguments are valid, we would expect a similar broadening of the neural iso-rate contour if the rate criterion were simply increased. Such a broadening of the FTC was not observed, at least over a 20 dB range of response criterion, as can be seen from the data of Fig. 9. Here the changes in the shape of the neural tuning curve produced by a change in the response criterion is contrasted with the changes produced by electrical stimulation. In Fig. 9a data from 5 animals obtained before and after electrical stimulation are pooled. The animals were chosen because the electrical stimulus produced threshold elevations on the tail of the FTC close to 20 dB. The threshold elevation at CF in these cases was about 50 to 60 dB. This exaggerated elevation at CF is emphasized in Fig. 9b in which the difference between the FTCs before and after electrical stimulation have been plotted. Fig. 9c presents pooled data from 4 normal animals without electrical stimulation, and

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shows the changes in the neural iso-rate tuning curve produced by increasing the response criterion from the normal 20 spike/s increase {defined as threshold) to a criterion equal to the firing rate produced by a 7 kHz stimulus presented 20 dB above threshold. This difference is emphasized by the average difference curve of Fig. 9d. Any change in tuning curve shape produced by this change in response criterion should mimic the changes produced by a simple 20 dB desensitization of the inner hair cells or primary afferent neurons. As can be seen from Figs. 9b and d, this change in response criterion produced a minimal broadening of the neural tuning curve, quite unlike the broadening produced by the electrical stimulation. It is therefore unlikely that a simple desensitization of the lHCs or the afferent synapse and nonlinear growth of organ of Corti vibration were responsible for the greater elevation at CF relative to the tail of the FTC.

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Fig. 9. The changes in the shape of the neural iso-rate tuning curve produced by current stimulation and an increase in the response criterion in normal animals. (a): Frequency threshold curves before ( + ) and immediately after (0) electrical stimulation using a 2 spike/s increase in firing rate as the response criterion (pooled data from 5 animals). The average tuning curves before and after the electrical stimulus are shown by the solid lines. (b): The difference between the pre- and poststimulatory data of (a). Note that the average change in the tuning curve shows a greater threshold elevation near CF than on the low-frequency tail. (c): The change in neural iso-rate tuning curves produced by a change in response criterion from 2 spike/s (+) to a rate equal to the firing rate produced by a 7 kHz tone 20 dB above threshold (0) (pooled data from 4 animals). The average tuning curves are shown by the solid lines. (d): The difference between the tuning curves of (c) showing the relatively small change in tuning curve shape with the change in response criterion.

Changes in single neuron rate-level functions The data presented in Fig. 10 illustrates the changes in the neural rate-level functions for CF stimulation produced by electrical stimulation for 10 mm. Results are from 3 typical animals. Before electrical stimulation the firing rate rose monotonically, and began to saturate for acoustic stimuli about 30 dB above threshold (Sachs and Abbas, 19’74). A short time after electrical stimulation (between 3 and 9 mm for the results of Fig. 10)

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Fig. 10. Neural rate-level functions for acoustic stimuli before electrical stimulation at the intensities indicated (10 min) and after partial recovery. The recovery times are indicated for each curve. The acoustic stimulus in each case was at the pre-stimulatory CF of each neuron. Electrical stimulation produced an elevation of threshold and a reduction in maximal firing rate.

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Fig. 11. Neural rate-level functions for acoustic stimulation on the tail of the frequency threshold curve (7 kHz). The CF of this neuron was 17 kHz. The curves were obtained before. immediately after and 39 min after electrical stimulation (100 pA, 10 min). Note the lack of threshold elevation and the marked fall in maximal firing rate immediately after electrical stimulation. After recovery the spontaneous and maximal firing rates were similar to their pre-stimulatory values.

threshold of the neurons was elevated, as indicated by a displacement of the curves to higher intensities, and the maximal firing rate was also reduced. In some animals both neural threshold and maximal firing rate recovered partially after periods from 25 to 50 min (Fig. 10). We have not characterized this recovery process in any detail. A similar fall in maximal firing rate was present for acoustic stimulation on the low-frequency tail of the FTC. In some cases of electrical stimulation this fall in maximal firing rate could be observed without an associated change in neural threshold on the tail. One example is presented in Fig. 11. Such examples serve to highlight the complexity of the changes that may be observed following electrical stimulation. Changes in the dynamic response properties of single neurons The complex changes in the cochlea produced by electrical stimulation are also evident in the changes in the dynamic response of the afferent neurons to tone burst stimuli. Peri-stimulus time histograms were collected before and after electrical stimulation. To avoid the complications associated with the complex changes at the tip of the FTC, these responses were collected using tail frequency stimulation (7 kHz). Since adaptation in

the single neuron firing rate can be observed over the first 200 ms or so of acoustic stimulation. peri-stimulus time histograms were collected during a 200 ms acoustic stimulus and for a further 50 ms following it. The period between two acoustic stimuli was 300 ms. The stimulus tone burst was presented at a level 40 dB above threshold. Fig. 12 illustrates the changes seen after electrical stimulation. In this case the cell had a CF of 17 kHz. Following electrical stimulation at 100 PA for 10 min the steady state firing rate was reduced, but the initial onset response was enhanced. After recovery (41 min) both the onset and steady state responses recovered to rates near their pre-stimulatory values.

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Fig. 13. SEM pictures of the guinea pig cochIea after electrical stimulation (900 PA). A: SEM picture of the first half of the basal turn of the guinea pig cochlea (s&e bar 300 pm). B: Magnification of the middle of the basal turn showing that no damage of the organ of Corti was visible at this level (scale bar 10 pm). C: Magnification of the damage in the first quarter of the basal turn. There was an absence of the microvilli at the apex of the supporting cells and a crumpled aspect of this surface. There was also some fusion of the stereo&a on OHCs (arrows) (scale bar 5 pm). D: High magnification of stereocilia fusion on OHC (scale bar 2 pm). E: High magnification of an example of total fusion of the hair bundle on an IHC (scale bar 2 pm). F: High magnification of partly fused stercocilia on an IHC (scale bar 2 pm).

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Histological

effects

Scanning electron microscopy (SEM) Histological damage was found only after very high current stimulation corresponding to currents three times that required to abolish the single unit action potential (about 900 PA). When damage was observed it was restricted to the first quarter of the basal turn (Figs. 13a and b). The surface of the organ of Corti was normal for regions more apical than the first half of the basal turn (Fig. 13b). In the damaged area an absence of microvilli at the apical surface of the supporting cells and a crumpled aspect of this surface was observed (Fig. 13d). Several hair cells showed stereocilia fusion (Figs. 13d, e and f). This damage was greater on the OHCs. On the IHCs the stereocilia fusion could be total (Fig. 13e) or partial (Fig. 13f), but these hair cells were always surrounded by IHC with undamaged stereocilia. Transmission electron microscopy (TEM) TEM examination revealed morphological changes for current levels as low as 300 PA (Figs. 14 and 15) mainly consisting of swollen mitochondria. In the IHCs the majority of mitochondria were swollen (Figs. 14a, b and c). This was most evident in the apical region of the IHCs, near the cuticular plate (Fig. 14b), and at the basal region of the cells, near the afferent synapse (Fig. 14~). The afferent dendrites also contained some swollen mitochondria (Fig. 14~). There were no swollen mitochondria in the OHCs (Figs. 15a, b and c). In the efferent endings beneath the OHCs, however, all mitochondria were swollen (Figs. 15a and b), and swelling was more voluminous than in the IHCs. Swollen mitochondria were also found in all unmyelinated regions of the efferent neurons (Fig. 15a). Discussion The changes produced in the cochlea by negative direct current injection into Scala tympani were complex. During electrical stimulation there was a fall in the amplitude of single unit action potentials and a transient increase in neural firing rate. This increase in firing rate has previously

been observed in the gross electrical activity of the auditory nerve (Schreiner et al., 1986). After stimulation single neuron threshold could be elevated at all ,frequencies across the FTC, although this was not always the case. In some cases the neural thresholds near CF were elevated with little or no change on the low-frequency tail of the FTC. In those cases in which tail elevation occurred the elevation near CF was greater. Similar changes in the tuning properties of the cochlea have been observed by others during and after electrical stimulation of the cochlea (Nuttall, 1985; Konishi et al., 1970; Teas et al., 1970; Hubbard et al., 1983). After electrical stimulation the maximal neural firing rate was reduced, the onset response of neurons to tone burst stimuli was enhanced. and the steady state firing rate decreased. It is unlikely that the changes we have observed were due to a heating of the cochlear structures by the passage of current. For example, a 500 PA current delivered into a 2 kS2 resistance, which approximates the access impedance of Scala tympani (Johnstone et al., 1966) would dissipate 0.5 mW of power. Over 10 mm this would deliver 300 mJ of energy to the cochlea. Without cooling this would raise the temperature of 50 ~1 of water (approximately cochlear fluid volume) by about 1.5 o C. This calculation takes no account of cooling by cochlear blood flow or normal thermal conductivity, which should reduce the temperature elevation markedly. Although the additional power dissipated across the electrode tip resistance would add to the heat flow into the cochlea, it seems unlikely that the heating effect was significant. Our interpretation of the complex changes we have observed is based on our present working model of cochlear transduction, which is summarized in Fig. 16. According to this view cochlear transduction can be divided into two major divisions, as delimited by the shaded areas. To the left of the figure are those processes that determine the mechanical response within the cochlea and produce the high sensitivity and frequency selectivity of the vibration of the organ of Corti. To the right are those processes that detect this vibration and control transmitter release and neural firing. For convenience we will call these two major divisions the ‘motor’ and ‘sensory’ components of cochlear transduction, respectively.

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15

33 we have observed. In some cases dramatic threshold elevations near CF were observed with little or no elevation on the tail (Fig. 6) and, even when tail elevation was present, the changes near CF were too large to be explained by the compressive growth of vibration near CF in the normal animal (Fig. 6, 8 and 9). For example, a 50 dB elevation at CF with less than 20 dB of elevation on the tail would require the input-output function of organ vibration at CF to have a slope of 0.4 dB/dB near neural threshold. Generally, this is not the case. Sellick et al. (1982) have observed slopes close to 1 dB/dB for basilar membrane vibration in the guinea-pig for at least the first 20 dB above neural threshold, while Robles et al. (1986) have observed slopes of about 0.5 dB/dB at CF in the chinchilla. Although the slopes observed by Robles et al. approach the required 0.4 dB/dB, this figure is an upper limit for the required nonlinear compression. There is also evidence from this and other studies that raising the response criterion for the FTC (Fig. 8, Geisler et al., 1974) or reducing transmitter release with MgZf perfusion (Siegel and Relkin, 1984) does not produce sufficient broadening of the FTC to explain the changes observed after electrical stimulation. The elevation of neural threshold in our experiments was therefore more likely to include a disruption of the motor processes within the cochlea, and hence a reduction in the vibration of the organ of Corti for frequencies near CF. Such a loss of mechanical sensitivity near CF has been observed after general cochlear deterioration and surgical trauma (Sellick et al., 1982; Robles et al., 1986) following drainage of perilymph (Patuzzi et al., 1982) and during the presentation of intense low-frequency tones (Patuzzi et al., 1984). A similar elevation of the neural FTC tip always occurs

In brief, stapes vibration induces intracochlear pressure fluctuations and transverse vibration of the cochlear partition. This motion stimulates the inner and outer hair cells of the organ of Corti by deflecting their hair bundles, changing the ionic conductance at the apical surface of these cells and producing receptor current through them. We assume that the OHC receptor current or its associated receptor potential controls an active force generation process, commonly termed the ‘active process’. The influence of the active forces, presumed to be generated by the OHCs, on the acoustical impedance of the organ of Corti enhances the sensitivity and frequency selectivity of the vibration of the organ of Corti near CF. This electromechanical feedback is commonly termed ‘negative damping’, and is shown in Fig. 16 as a feedback loop from the force generation stage of transduction to the micromechanical motion of the organ of Corti (Neely and Kim, 1983, 1986). The transmembrane voltage across the basolateral walls of the IHCs, which is normally dominated by the intracellular receptor potential, controls transmitter release and finally neural firing rate. Changes in the neural FTC may be produced by disruption of one or more of these stages of transduction. Changes in the sensory processes of transduction are likely to produce elevations of the FTC at all frequencies. It is unlikely that threshold elevation would be equal at all frequencies, however. Although changes to the sensory processes of transduction would not alter the tuned vibration of the organ of Corti, the compressive growth of the vibration of the organ of Corti near CF should produce greater threshold elevation near CF than on the tail of the FTC. as described in the results section of this report. As already outlined, this explanation cannot explain the range of changes

Fig. 14. TEM pictures of an IHC after electrical stimulation (300 PA). A: All mitochondria in the IHC are swollen (scale bar 3 pm). B: High magnification of the apex of an IHC showing swelling of mitochondria (scale bar 1 pm). C: High magnification of the basal part of an IHC showing swollen mitochondtia in the IHC and also the presence of small swelling in an afferent dendrite (scale bar 1 pm). Fig. 15. TEM pictures of OHCs and efferent neurons after electrical stimulation (300 PA). A: The mitochondria m the OHCs were normal, but there was voluminous swelling of mitochondria (arrow) in the efferent neurons (scale bar 2 pm). B: High magnification of the apical part of the OHC showing that all mitochondria were normal (scale bar 1 pm). C: High magnification of an efferent ending showing voluminous swelling of mitochondria (scale bar 0.5 pm).

34

r-4 STAPES

MOTOR PROCESSES

DlSPLACEMENl

HYOKIDYNAWX

1984), and after kanamycin treatment (Robertson and Johnstone, 1979b). It has also been shown that intense low-frequency tones (Patuzzi and Sellick, 1984), efferent activity (Galambos. 1956: Wiederhold, 1970; Wiederhold and Kiang, 1970) and acetylcholine perfusion (Robertson and Johnstone, 1978) can induce changes of the FTC tip without significant changes for the tail frequencies. These and other experiments suggest that the integrity of the OHCs is necessary for normal frequency-selectivity, probably because they are necessary for the normal vibration of the organ of Corti. On the basis of these arguments the complex changes in the FTC produced by electrical stimulation in this study were probably the result of a combination of effects on the sensory and motor processes of transduction. Certainly, the changes we have observed cannot be explained by a simple depletion of transmitter from the IHCs as originally hypothesized. Such a depletion would not have elevated the tip of the FTC without elevating the FTC tail. The mechanism of tip elevation

Fig. 16. The processes in cochlear transduction can be divided into two broad categories. Those processes associated with the outer hair cells that appear to enhance the vibration of the organ of Corti may be termed ‘motor processes’. Those processes associated with the inner hair cells and release of the afferent neuro-transmitter act to detect this vibration, and may be termed ‘sensory processes’. Changes in the neural tuning curve may arise from disruption of any of these processes.

in temporary threshold shift following loud sound (Cody and Johnstone, 1980), often following chronic acoustic trauma (Liberman and Dodds,

Liberman and Dodds (1984) have shown a correlation between disruption of the hair bundles of OHCs and loss of the tip of the FTC following chronic acoustic overstimulation. In our study such disruption was not present, except when current intensities 3 times greater than that necessary to produce threshold elevation were used. It is evident from other experiments, however, that gross anatomical disruption of the hair bundles is not a necessary condition for threshold elevation near CF. For example, the tip of the neural FTC can be cyclically elevated in a normal cochlea by an intense low-frequency tone (Patuzzi and Sellick, 1984; Patuzzi et al., 1984). Similar reversible changes are seen after drainage of perilymph (Robertson, 1974; Patuzzi et al., 1982) and after acute acoustic overstimulation lasting only minutes (Cody and Johnstone, 1980). In all these instances there is no significant anatomical correlate to the tip elevation (e.g. Robertson, 1983; Liberman and Dodds, 1984). It is known, however, that during modulation with low-frequency tones and following acoustic trauma that there is a significant

impairment of OHC mechano-electric transduction process, as indicated by a fall in the low frequency microphonic from the basal turn (Patuzzi et al., 1987), or modulation of the AC receptor currents in the organ of Corti (Patuzzi and Sellick, 1984). A similar disruption of the OHC mechano-ele~t~c transduction was suggested in this study by the fall in the low-frequency microphonic following current stimulation (Fig. 4). Since no anatomical disruption was observable in TEM or SEM, the processes affected by the current were probably at the molecular level. We may hypothesize, then, that tip elevation in these experiments is an analogue of temporary threshold shift following acoustic overstimulation. While such a disruption of the OHC mechanoelectric transduction mechanism is, in our view, the most likely explanation for elevation of the FTC tip, it is also plausible that the current stimulation disrupted the electro-mechanical force generation stage of the motor process (Brownell, 1983; Brownell et al., 1985; Ashmore, 1986; Neely and Kim, 1986). Althou~ there seemed no obvious disruption of OHC structures at the TEM level, swelling of mitochondria in the efferent endings beneath OHCs was observed. Since swelling of mitochondria is normally associated with elevated intracellular levels of Ca*+ (Hackenbroch, 1968; De Robertis and De Robertis, 1980) which they sequester (Bygrave, 1978). and elevated Ca2” is associated with increased electrical activity of neurons (Baker, 1972; 1976; Baker et al., 1971) the swollen mitochondria may indicate an accelerated release of efferent neurotransmitter. It is possible that direct electrical stimulation of the efferent terminals with a consequent release of neurotransmitter elevated the tip of the FTC (Galambos, 1956; Wiederhold, 1970; Wiederhold and Kiang, 1970; Robertson and Johnstone, 1979b). It is certainly known that electrical stimulation at the round window can produce some effects associated with efferent stimulation (Rajan and Johnstone. 1983). The effect of such release of the efferent neurotransmitter are not normally longlasting, however, and are also associated with an increase in the cochlear ~cropho~c (Fex, 1959), and threshold elevation of less than 30 dB. Hence, the reduction of the low-frequency microphonic observed in this study (Fig. 4) is not consistent

with a simple long lasting effect of the efferent fibres on OHC function. It is possible that electrical stimulation produced both a reduction in the mechano-electric transduction-electric transduction sensitivity of the OHCs and a disruption of the active process by efferent stimulation, but if this were the case then the relative contribution from each effect to the threshold elevation at CF would be difficult to establish.

Although these arguments exclude a desensitization of the sensory processes of transduction as the sole effect of electrical stimulation, the elevation of threshold on the FTC tail and the complex changes in the neural rate-level curves and peristimulus time histograms indicate that changes in the sensory processes of transduction may have occurred. There are, however, at least 5 possible explanations for an elevation of the FTC tail. These are: (i) A change in the vibration of the organ of Corti at low frequencies. (ii) A reduction in the sensitivity of IHC mechano-electric transduction process. (iii) Prolonged cathodic block of the afferent dendrites. (iv) A block of post-synaptic receptors on the afferent dendrite due to either an excess of neurotransmitter or a direct effect of electrical stimulation. (v) A presynaptic exhaustion of neuro-transmitter or a disruption of the transmitter release process. As already mentioned, it is unlikely that a reduction in the amplitude of vibration of the organ of Corti explains the tail elevation. The vibration of the organ of Corti for tail frequency stimulation seems extremely robust after a variety of manipulations of the cochlea, including loud sound, drainage of perilymph, surgical trauma and even death (Sellick et al., 1982; Patuzzi et al.. 1982). Moreover, a simple fall in the mechanical stimulus to the IHCs would not produce a fall in the maximal firing rate of the afferent neurons (Figs. 10 and 11). If there was a change in the vibration of the organ of Corti it must have been

36

accompanied by other changes in either the IHCs or the afferent neurons. We carmot exclude disruption of IHC mecharm-electric transduction as the cause of tail elevation and the fall in the maximal firing rate of the neurons. Certainly OHC mechano-electric transduction was impaired by the electrical stimulation, as indicated by the fall in the round window ~cropho~c (Fig. 4). However, the changes observed in the peri-stimulus time histograms following electrical stimulation are not consistent with a simple fall in the amplitude of the IHC receptor potential. Such a reduction would produce a fall in the steady-state and onset responses of the afferent neuron, similar to that seen when an acoustic stimulus is reduced in intensity (Kiang, 1962). Neither p change in the mechanical drive to the IHC nor a reduction in IHC sensitivity would be expected to produce an increase in the onset response of the neuron. Hence, at least some of the changes observed were probably associated with changes at the IHC/primary afferent synapse. The elevation in the tail of the FTC could possibly be due to a direct influence of the current on the afferent dendrites. That the current does have an effect on the dendrites is indicated by the fall in the amplitude of the single neuron action potential during stimulation. This decrease is similar to cathodic block in the giant axon of Loligo (Hodgkin and Huxley, 1952) and in myelinated nerve fibres of toad (Fr~enhauser and Persson, 1957; Woodbury, 1966). In these preparations a constant depolarization of a neuron by extracellular current injection can induce an inactivation of the axon in the region of the stimulating electrode. For example, in the giant axon of Loligo, a steady depola~sation of 10 mV can induce a 60% decrease of the action potential amplitude, and a 20 mV depolarization can cause total suppression (Hodgkin and Huxley, 1952). Although the suppression of the action potential does indicate a direct effect of the current on the afferent dendrite, on the basis of our data we are unable to discriminate between changes in neural threshold produced by cathodic block and those produced by other mechanisms, such as blockage of the receptors on the post-synaptic membrane or disruption of the pre-synaptic release of trans~tter.

Effect on single neuron firing rate without acoustic stimulation The mechanism by which the negative current stimulation produces the transient increase in neural firing during stimulation is not clear. This increase in neural firing is consistent with the results of Schreiner et al. (1986) who described the same effect on the gross spontaneous activity of the auditory nerve, and with the single neuron experiments of other authors (Teas et al., 1970; Konishi et al., 1970). Although the effects reported during electrical stimulation appear consistent from study to study, there is no consensus on the mechanism producing the increase in neural activity. Teas et al. (1970) have proposed that the increase in firing rate may be due to an alteration of the post-synaptic membrane potential of the afferent dendrites. The fact that the action potential amplitude decreased during the stimulation indeed shows that the afferent dendrites were affected by the current. On the other hand, Schreiner et al. (1986) have suggested that electrical stimulation produces a depolarization of the basolateral walls of the IHCs, with a consequent release and depletion of neurotrans~tter. The presence of swollen mitochondria in the IHCs in this study possibly reflected this excitation. These observations suggest that current injection produces both pre- and post-synaptic effects, although they do not allow an estimate of the relative cont~bution of each to the increase in firing rate at the start of electrical stimulation, nor to the changes observed after it. The loss of spontaneous firing immediately after electrical stimulation that abolished the action potential may also be due to pre- or post-synaptic changes. It is known that following prolonged direct current stimulation of the squid axon that action potentials cannot be evoked by electrical stimulation for some time afterward (Rudy, 1978). This has been attributed to inactivation of sodium channels. Similarly, the rapid decrease of the firing rate during electrical stimulation may have resulted from a blockage of the post-synaptic receptors due to an excess of neurotransmitter (Bobbin, 1979), or have been due to an exhaustion of neurotransmitter reservoirs within the IHCs, as suggested by

31

Schreiner et al. (1986). Although Bobbin’s experiments showed that a perfusion of glutamate induced an increase followed by a fast decrease of firing rate, a post-synaptic excess of neurotransmitter is often associated with voluminous swelling of the afferent dendrites (Robertson, 1983; Goulios and Robertson, 1983; Pujol et al., 1985). In our experiments only a few swollen mitochondria were found in the primary afferent neurons, and no swelling of the afferent dendrites was observed. This suggests that the post-synaptic effects produced by an overabund~ce of neurotransmitter were relatively small, although the lack of swelling of the afferent dendrites may simply have been due to the short duration of the current stimulus.

Locaiization cations

of the current effects and clinical impli-

The electrical stimulation induced elevation of the CAP threshold only at frequencies higher than 10 kHz, and the maximum elevation was a function of the stimulating electrode position. These results are in accord with those of Schreiner et al. (1986). Tasaki and Femandez (1952) obtained similar results passing current between two electrodes positioned in Scala tympani and Scala vestibuli. They showed that a current injected in the basal turn has no effect in the apical turns. An opposite result was found by Van den Honert and Stypulkowski (1984). Using 100 ps pulses at 25/s, they showed that the entire cochlea was affected by the current. This difference may be due to the very short pulses used and the different capacitive pathways for AC current within the cochlea. In any case the duration of current stimulation seems important in determining the spatial extent of electrical stimulation. Finally, these experiments have some relevance to the clinical treatment of tinnitus in human subjects (Cazals et al., 1978; Portman et al,, 1979; Aran, 1981; Aran and Canals, 1981). If the area affected by the current used to induce a suppression of the tinnitus is small, as in our experiments, the cells producing the tinnitus must be near the electrode. If these cells are too remote, the current must be increased. As we have described, high

current can produce hair cell lesions and loss. Even if the electrical stimulation good therapy for tinnitus, more detailed the effects of the pulse length should give ing results.

heating seems a study of interest-

Conclusions These experiments have shown that negative direct current stimulation of Scala tympani of the basal turn of the guinea pig cochlea produces complicated changes in the neural response of primary afferent neurons. Gross morphological disruption of the hair cells was not the cause of the changes observed. The fact that threshold elevation near CF could occur without changes on the FTC tail suggests that normal vibration of the organ of Corti could be disrupted by current stimulation, without a desensitization of either the IHCs or the afferent neurons themselves. This was probably due to a disruption of OHC mechanoelectric transduction-transduction. The changes on the FTC tail, which included neural threshold elevation, reduction of maximal firing rate and changes in the dynamic response of neurons to tone bursts, indicate that processes other than the vibration of the organ of Corti were also affected by electrical stimulation. Whether these changes were due to complex changes in the storage and release of the afferent neuro-transmitter substance as originally hypothesized is still unclear. If direct current stimulation of the cochlea is to be used in the investigation of the inner hair cell/primary afferent synapse a more detailed understanding of these complex changes will be required. Acknowledgements The authors wish to acknowledge K.S. Cole and IS. Corbett for technical assistance, Drs. G.K. Yates and D. Robertson for criticizing the manuscript. These studies were supported by the National Health and Medical Research Council and the University of Western Australia. References Aran, J.M. (1981) Electrical stimulation of the auditory system and tinnitus control. Br. J. Laryngol. Otol. 95. 153-162.

3x Aran. J.M. and Cazals. Y. (1981) Electrical suppression of tinnitus. In: Tinnitus, A Ciba Foundation Symposium. Pitman, London, pp. 217-231. Ashmore. J.F. (1986) A fast motile response in guinea pig outer hair cell: The cellular basis of the cochlear amplifier. J. Physio!. 388. 323-347. Baker, P.F. (1972) Transport and metabolism of calcium ion in nerve. Prog. Biophys. Molec. Bio!. 24, 177-223. Baker. P.F. (1976) The regulation of intracellular calcium. Symp. Sot. Exp. Bio!. 30, 67-88. Baker. P.F., Hodgkin, A.L. and Ridgway. E.B. (1971) Depolarization and calcium entry in squid giant axons. J. Physiol. 218, 709-755. Bobbin. R.P. (1979) Glutamate and aspartate mimic the afferent transmitter in the cochlea. Exp. Brain Res. 34. 389-393. Brownell, W.E. (1983) Observations of a motile response in isolated outer hair cells. In: W.R. Webster and L.M. Aitkin (Eds.). Mechanisms of Hearing, Monash llniversity Press. Clayton, Australia. pp. 5-10. Brownell. W.E.. Bader, CR., Bertrand, D. and de Ribaupierre, Y. (1985) Evoked mechanical responses of isolated cochlear outer hair cells. Science 227. 1944196. Bygrave, F.L. (1978) Mitochondria and the control of mtracellular calcium. Bio!. Rev. 53, 43-79. Cazals, Y.. Negrevergne, M. and Aran, J.M. (197X) Electrical stimulation of the cochlea in man: hearing induction and tinnitus suppression. J. Am. Audio!. Sot. 3. 209-213. Cheatham. M.A. and Dallas, P. (1982) Two-tone interactions in the cochlear microphonic. Hear. Res. 8. 29-48. Cody, A.R. and Johnstone, B.M. (1980) Single auditory neuron response during acute acoustic trauma. Hear. Rez. 3. 3-16. Fex, J. (1959) Augmentation of cochlear microphonics by stimulation of efferent fibres to the cochlea. Acta Oto!atyngo!. 50, 540-541. Frankenhauser. B. and Persson, A. (1957) Voltage clamp experiments in myelinated nerve fibre. Acta Physiol. Stand. 42, Supp!. 145, 45. Galambos, R. (1956) Suppression of auditory nerve activity by stimulation of efferent fibres to cochlea. J. Neurophysio!. 19. 424-437. Goulios, H. and Robertson, D. (1983) Noise-induced cochlear damage assessed using electrophysiological and morphological criteria: an examination of equal energy principle Hear. Res. 11. 327-341. Hackenbrock. C.R. (1968) Ultrastructural basis for metabolically linked mechanical activity in mitochondria. I!. Electron transport-linked ultrastructural transformatton in mitochondria. J. Cell. Bio!. 37, 345-369. Hodgkin, A.L. and Huxley, A.F. (1952) The dual effect of membrane potential on sodium conductance in the giant axon of Lohgo. J. Physio!. 116, 497-506. Hubbard, A.E.. Voight, H.F. and Mountain. D.C. (1983) Injection of direct current into scala media alters auditory nerve response properties. Assoc. Res. Otolaryngol. Abstr. 145. pp. 103-104. Kiang. N.Y-S. (1962) Discharge patterns of single fibres in the cat’s auditory nerve. Research Monograph 35. M.I.T. Press. Cambridge. Massachusetts.

Konishi, ‘I‘., Teas, D.C. and Wemick. J.S. (1970) Effects of electrical current applied to cochlear partition on dtscharge in Individual auditory-nerve fibrrs. I. Prolonged direct-current polarization. Acoust. Sot. Am. 6. 1519 1526. Liberman. M.C. and Dodds, L.W. (1984) Single neuron lahelling and chronic cochlear pathology. III. Steretrilta damage and alteration of threshold tuning curves. Hear. Res. 16. 55- 74. Neely, ST. and Kim. D.O. (1983) An active cochlear model showing sharp tuning and high sensitivity. llcar. Re\. 9. 123-130. Neely. ST. and Kim, D.O. (1986) A mode! for active elements in cochlcar biomechanics. J. Acoust. Sot. Am. 79. 1472.-1480. Nutta!!, A.L. (1985) influence of direct current on dc receptor potentials from cochlear inner hair cells in the guinea pig. J. Acoust. Sot. Am. 77, 165-175. Patuzzi, R. (1987) A mode! of the generation of the cochlear microphonic with nonlinear hair cc!! transduction and nonlmear basilar membrane mechanics. Hear. Res. 30. 73--82. Patuzzi. R.B. and Sellick. P.M. (1984) The modulation of the sensitivity of the mammalian cochlea by low frequency tones. I!. Inner hair cell receptor potentials. Hear. Res. 13. 9-18. Patuut. R.. Sellick, P.M. and Johnstonc. B.M. ( 1982) Cochlear drainage and basilar membrane tuning. J. Acoust. Sot. Am. 72. 10641065. Patuzzi, R.B.. Sellick. P.M. and Johnstone. B.M. (1984) The modulation of the sensitivity of the mammalian cochlea by low frequency tones. III. Basilar membrane motion. Hear. Res. 13, 19-27. Patuzzi. R.B., Yates. G.K. and Johnstone. B.M. (1987) Notse trauma and outer hair cell Impairment (abstr.). In: Proc Workshop on Inner Ear Biology. 24th. Nijmegen. The Netherlands, p. 44. Portmann. M.. Cazals. Y.. Negrevergne. M. and Aran. J.M. (1979) Temporary tinnitus suppression in man throufi electrical stimulation of the cochlea. Acta Otolaryngo!. X7. 294-299. Pujol. R.. Lenoir. M., Robertson. D.. Eybalin. M. and Johnstone. B.M. (1985) Kainic acid selectively alters auditory dendrites connected with cochlear inner hair cells. Hear. Res. 18, 145-151. RaJan, R. and Johnstone, B.M. (1983) Efferent effects ehcitcd by electrical stimulation at the round window of the guinea pig. Hear. Res. 12, 405-417. Robertis. de E. and Robertis. de EM. Jr. (1980) Mitochondria Jnd oxidativc phosphorylation. In: Cell and Molecular Biology. Saunders College. Philadelphia. pp. 249-281. Robertson. D. (1974) Cochlear neurons: frequency selectivity altered by perilymph removal. Science 186, 623-628. Robertson, D. (1983) Functional significance of dendrittc swelling after loud sounds in the gumea pig cochlea. Hear. Res. 9. 263-278. Rohertson, D. (1984) Horseradish peroxidase inJection of physiologically characterized afferent and efferent neurons in the guinea pig spiral ganglion. Hear. Res. 15, 113-122. Robertson. D. and Manley. GA. (1974) Manipulation of

39 frequency analysis in co&ear ganglion of the guinea pig. J. Comp. Physiol. 91, 363-375. Robertson, D. and Johnstone, B.M. (1978) Efferent transmitter substance in the mammalian cochlea: single neuron support for acetylcholine. Hear. Res. 1. 31-34. Robertson. D. and Johnstone, B.M. (1979a) Effect of divalent cation on spontaneous and evoked activity of single mammalian auditory neurons. PfIugers Arch. 380, 7-12. Robertson. D. and Johnstone, B.M. (1979b) Aberrant tonotopic organization in the inner ear damaged by kanamycin. J. Acoust. Sot. Am. 66, 466-469. Robles. L., Ruggero, M.A. and Rich, N.C. (1986) Basilar membrane mechanics at the base of the chinchilla cochlea. I. Input-output functions. tuning curves, and phase responses. J. Acoust. Sot. Am. 80, 1364-1374. Rudy, B. (1978) Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance. J. Physiol. 283, Sachs. M.B. and Abbas, P.J. (1974) Rate versus level functions for auditory-nerve fibres in cats tone-burst stimuli. J. Acoust. Sot. Am. 56, 1835-1847. Schreiner, C.E., Snyder, R.L. and Johnstone, B.M. (1986) Effects of extracochlear direct current stimulation on the ensemble auditory nerve activity of cat. Hear. Res. 21, 2133226. Sellick, P.M., Patuzzi, R.B. and Johnstone, B.M. (1982) Measurement of basilar motion in the guinea pig using the Miissbauer technique. J. Acoust. Sot. Am. 72, 131-141. Sewel. W.F. (1984) The effects of furosemide on the endo-

cochlear potential and auditory-nerve fiber tuning curves in cats. Hear. Res. 14, 305-314. Tasaki. I. and Fernandez. C. (1952) Modificatton of cochlear microphonics and action potentials by KCI solution and by direct current. J. Neurophysiol. 15. 497-512. Teas. D.C., Konishi, T. and Wernick. J.S. (1970) Effects of electrical current applied to cochlear partition on discharges in individual auditory-nerve fibres. II. Interaction of electrical polarization and acoustic stimulation. J. Acoust. Sot. Am. 6, 1527-1537. Van den Honert. C. and Stypulkowski. P.H. (1984) Physiological properties of the electrically stimulated auditory nerve: 11. Single fibre recordings, Hear. Res. 14, 2255243. Whitfield. I.C. and Ross. H.F. (1965) Cochlear microphonic and summating potentials and the outputs of individual hair cell generators. J. Acoust. Sot. Am. 38, 1266131. Wiederhold, M.L. (1970) Variations in the effects of electric stimulation of the crossed olivocochlear bundle on cat single auditory-nerve-fiber responses to tone bursts. J Acoust. Sot. Am. 48, 966-977. Wiederhold. M.L. and Kiang, N.Y-S. (1970) Effects of electric stimulation of the crossed olivocochlear bundle on single auditory-nerve-fibres in cat. J. Acoust. Sot. Am. 48, 950-965. Woodbury, J.W. (1966) Action potential: Properties of excitable membranes. In: T.C. Ruth and H.D. Patton (Eds.). Physiology and Biophysics. W.B. Saunders Company. Philadelphia and London, pp. 26658.