Recovery of structure and function of inner ear afferent synapses following kainic acid excitotoxicity

Itmlll¢, ELSEVIER

Hearing Research 105 (1997) 65 76

Recovery of structure and function of inner ear afferent synapses following kainic acid excitotoxicity Xiang-Yang Zheng, Donald Henderson *, Bo-Hua Hu, Sandra L. McFadden Hearing Research Laboratories, 215 Parker Hall, Department o f Communicative Disorders and Sciences, State University of New York at Buffalo, Buffalo, N Y 14214, USA Received 5 July 1996; revised 11 October 1996; accepted 26 October 1996

Abstract

The present study was conducted to examine the re-establishment of IHC/VIII nerve synapses following kainic acid (KA) excitotoxicity and to discern if the re-organized afferents could render not only a normal auditory threshold but also a normal suprathreshold function. KA (60 mM) applied to the intact round window membrane in chinchilla destroyed postsynaptic endings of the auditory nerve, depressed the input-output (I/O) functions of auditory evoked potentials (EVP) and produced an average loss of sensitivity of over 80 dB at 4, 8, and 16 kHz, with less substantial losses (40-60 dB) at lower frequencies. However, there was no significant difference in 2fl f2 distortion-product otoacoustic emissions (DPOAE) before and after the application of KA. The nerve endings went through a sequence of swelling, degeneration and recovery over a 3-5 day period at higher frequency. Auditory sensitivity and supra-threshold response returned accordingly. In contrast, complete recovery at lower frequencies (1 and 2 kHz) required more than 5 days. The results provide strong evidence that (1) excitotoxically damaged cochlear afferent neurons can recover and render both a normal EVP threshold and EVP I/O function and (2) afferent innervation to IHCs is not necessary for DPOAE generation.

Keywords." Cochlear nerve; Drug effect; Glutamate neurotoxicity; Hearing loss; Cochlea, base vs. apex

I. Introduction

A m o n g the probable neurotransmitters at the inner hair cell ( I H C ) / V I I I nerve synapse in the m a m m a l i a n cochlea, glutamate is considered the most likely candidate (Altschuler et al., 1989; Felix and Ehrenberger, 1990; Lefebvre et al., 1991; N a k a g a w a et al., 1991; Li et al., 1994). G l u t a m a t e and its analogues can have negative side effects related to their excitatory properties when these amino acids are released in excess or incompletely recycled. In several studies with guinea pigs, rats and chinchillas, artificial perilymph containing kainic acid (KA) at various concentrations was either perfused through the scala tympani (Bledsoe et al., 1981; Juiz et al., 1989), injected through the round window (Pujol et al., 1985), or directly applied to the * Corresponding author. Fax: +1 (716) 829 2980; e-mail: [email protected]

0378-5955 / 97 / $17.00 © 1997 Elsevier Science B.V. All rights reserved P11S0378-5955(96)00188-8

intact round window m e m b r a n e (Dolan et al., 1990; Zheng et al., 1996). In all cases, the findings were strikingly similar in that K A application reduced or abolished cochlear nerve action potentials (CAP) but had little, if any, effect on cochlear microphonics. Collectively, these studies provide evidence that K A exerts a selective excitotoxic action on afferent nerve fibers, resulting in swelling and disruption of type I dendrites. These effects are comparable to what acutely occurs after acoustic trauma or severe cochlear ischemia (Spoendlin, 1971; Robertson, 1983; Pujol et al., 1993). Both K A and o~-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) are analogs of glutamate. Studies have shown KA-related neurotoxicity to be irreversible, leading to neuron degeneration and death in the nervous system (Coyle, 1983; R o t h m a n and Olney, 1987; Janssen et al., 1991). F o r example, the abolished CAP as a result of administration of a 10 nmol dose of K A appeared to be irreversible (Bledsoe et al., 1981)

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X..-E Zheng et al./Hearing Research 105 (1997) 65 76

and 5 nM KA caused a loss of spiral ganglion neurons examined after a 10 day survival time (Juiz et al., 1989). However, repair of auditory afferent nerve synapses in mammals after an excitotoxic injury following AMPA treatment has been demonstrated by Pujol et al. (1995). In their study, local application of 200 m M AMPA immediately resulted in the destruction of all postsynaptic endings of the auditory nerve and a total loss of auditory brainstem responses. Five days after this excitotoxic injury, the IHCs were contacted by regenerating postsynaptic dendrites and the auditory brainstem responses had recovered. The discrepancies between the results from KA excitotoxicity studies (Bledsoe et al., 1981; Juiz et al., 1989) and the study of A M P A excitotoxicity (Pujol et al., 1995) could be explained in many ways. For example, it might be possible that KA-induced cochlear excitotoxicity is somehow different from that from A M P A treatment and renders the cochlear injuries to be irreversible. Another obvious hypothesis to explain the discrepancies is that it could be possible that only part of the AMPA-injured afferent synapses regenerated in the study of Pujol et al. A subpopulation of VIII nerve fibers could still render a normal ABR threshold, with deficits only reflected in the input/output (I/O) functions of auditory potentials. In an attempt to resolve these discrepancies, this study was designed (1) to determine if auditory nerve dendrites damaged by KA could recover and, if yes, to investigate if the re-organized auditory nerve dendrites could render a supra-threshold auditory evoked response as well as a normal auditory sensitivity, and (2) to discern the influence of IHC/VIII nerve fiber on the generation of 2f1-f2 distortion-product otoacoustic emissions (DPOAE), a noninvasive measure of the status of the outer hair cell (OHC) system (Lonsbury-Martin and Martin, 1990; Hall et al., 1994; Bergman et al., 1995).

2. Methods

2.1. Subjects and surgical procedures Seventeen healthy, adult chinchillas (450-650 g) of both sexes served as subjects. Each animal was anesthetized with a subcutaneous injection of ketamine (36 mg/ kg) and acepromazine (0.56 mg/kg) and made monaural by surgical destruction of the left cochlea. A chronic recording electrode (0.9,-- 1.1 cm long) was then stereotaxically implanted in the left inferior colliculus (Henderson et al., 1973). The reference electrode (0.4,-,0.5 cm long) was implanted in the rostral portion of the parietal bone. The surgery was performed at least 2 weeks prior to electrophysiological testing. The success of cochlear destruction was confirmed by demonstrating a complete absence of DPOAEs in the destroyed ear 2 weeks after the surgery.

For application of KA, each subject was again anesthetized with a subcutaneous injection of ketamine (36 mg/kg) and acepromazine (0.56 mg/kg). A postauricular incision was made to expose the cochlear part of the bulla. A hole of approximately 2 mm was made in the dorsal bulla, through which KA (60 m M in lactated ringers) was locally applied to the intact round window membrane using a syringe with a 31-ga needle. Care was taken to make sure that KA covered the round window membrane, but did not overflow into the middle ear cavity. The K A was left on the round window membrane for 30 min and then removed with small cotton points.

2.2. Recording and data collection procedures I/O functions of evoked potentials (EVP) and DPOAEs were measured before, then 3 h, and 1, 2, 3, 5 and 10 day(s) after K A treatment.

2.2.1. Evoked potentials Awake chinchillas were placed in a yoke-like harness to keep the animals' heads in a fixed position within the calibrated sound field for EVP testing. Test stimuli consisted of tone pips (alternate starting phase; 5 ms rise/ fall; Blackman ramp; 10/s) at frequencies from 1 to 16 kHz at octave intervals. The signals were generated digitally (16 bit D/A converter, 100 kHz sampling rate, 20 kHz low-pass filtered) by a digital signal processing board (Spectrum Signal Processing TMS320C25) located in a personal computer. The signal was routed through computer-controlled attenuator (127.5 dB, 0.5 dB step), buffer amplifier and then to the loudspeaker (Realistic 401197), located at a distance of approximately 38 cm in front of the animal's head. The electrical activity from the electrodes was amplified (20,000x), filtered (10-3000 Hz) and fed to an A/D converter (100 kHz, 1 6 bits) on a signal processing board. One hundred samples were averaged at each level and the level was varied in 5 dB steps from a sound level below threshold to well above threshold. The mid-point between the lowest level at which there was a clear response and the highest level at which there was no response was considered the threshold. 2.2.2. Distortion-product otoacoustic emissions DPOAEs were recorded at the primary frequencies (f2) at 1, 2, 4 and 8 kHz while the animals were lightly restrained (Subramaniam et al., 1994). All the stimuli were generated using D/A converters (16 bits, 100 kHz) on two separate signal processing boards located in a personal computer. The output of each channel was low-pass filtered (rolloff 90 dB between 20 and 24 kHz) and then sent to a computer-controlled attenuator, buffer amplifier, and sound source (Etymotic ER2) which was coupled to the microphone through a nar-

X.-K Zheng et al./Hearing Research 105 (1997) 65 76

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row tube. DPOAEs were measured with a low-noise microphone (Etymotic ER10B). The output of the microphone was lead to the A/D converter (16 bit) on a separate signal processing board and sampled for 500 ms at a sampling rate of 31 kHz. D P O A E I/O functions were recorded in 5 dB steps from 0 to 70 dB SPL. The levels of fl and f2 were equal and the f2/fa ratio was 1.2. For each I/O recording, eight time averages were used and samples were rejected from the average if the noise level exceeded 10 dB SPL.

2.3. Histopathological analysis Cochleae were analyzed immediately (from 30 min to 3 h) after K A exposure (5 ears) and when the auditory

sensitivity had recovered by approximately 20 ~ 30 dB (4 ears), 50 ,~ 60dB (4 ears) or 70 ,-~ 80 dB (4 ears). Each animal was anesthetized with sodium pentobarbital (1.0 ml, i.p.) and decapitated. The right bulla was then removed and prepared for light microscopy. The left bulla was examined to verify the destruction of the cochlea. The right cochlea was first perfused with 2.5% glutaraldehyde in phosphate buffer (pH 7.3-7.4) through the round window membrane for 2 h, then post-fixed with 1% OsO4 in phosphate buffer for 1 h. Segments (2-3 mm) of basilar membrane were removed from the cochlea ( ~ 3 mm from the base), dehydrated in ascending concentrations of ethyl alcohol (70%, 90% and 100%), 100% ethyl alcohol and acetone (1 : 1), 100% acetone, with 15 min duration for each change, and

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Fig. 3. Mean D P O A E I/O functions before (Pre, n = 17), 3 h (3h, n = 12) and 5 days (5d, n = 4 ) after K A treatment, measured at primary frequencies (f2) of 1, 2, 4 and 8 kHz. Hatched area shows the 95% confidence intervals around the mean values measured before K A application.

)L-E Zheng et al./Hearing Research 105 (1997) 65 76

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Fig. 4. Confocal microscopic image from a normal cochlea, showing inner hair cell (IHC), type I afferent endings (AE), inner pillar cell (IPC), outer pillar cell (OPC), tunnel of Corti (TC) and inner sulcus (IS). Bar = 5 gin.

All procedures involving care and handling of the chinchillas were reviewed and approved by the University Institutional Animal Care and Use Committee.

3. Results

3.1. E V P threshold shifts A perspective on the hearing threshold shifts at all the tested frequencies after K A exposure is provided in Fig. 1. Three hours after the K A application, hearing losses at various frequencies were substantial. There was an average hearing loss of about 80 dB at 4, 8 and 16 kHz, while the mean threshold shifts were around 40 dB at 1 kHz and 60 dB at 2 kHz. Variability was much greater at low than at high frequencies. Hearing recovered rapidly at higher frequencies (4, 8 and 16 kHz). It had recovered by about 50 dB by 3 days after KA treatment and recovered completely 2 days later. At the lower frequencies (1 and 2 kHz), hearing also

recovered, but at a slower rate. An overall 5-factor multiple regression, using the five frequency groups as independent variables showed that there were no significant differences (P > 0.05) in the rate of improvement in threshold data between either 1 and 2 kHz groups or amongst 4, 8 and 16 kHz groups. Thus, average thresholds for low (1 and 2 kHz) and high (4, 8 and 16 kHz) frequency stimuli were computed for analysis. Rate of recovery is compared in the bottom-right panel in Fig. 1. To avoid bias in estimating the recovery slopes, only data from ears with more than 60 dB hearing loss as measured 3 h after K A treatment and presumably with complete perfusion of KA were included in the regression analysis at each frequency. It is clear that recovery at high frequency (4, 8 and 16 kHz; 20 dB/day) was substantially more rapid ( P < 0.001) than recovery at low frequency (1 and 2 kHz; 9 dB/day) during the first 3 day period. After 5 days recovery, there was still a hearing threshold shift of about 5 dB at 1 and 2 kHz. Within the next 5 days, low-frequency hearing also recovered completely.

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AL-Y. Zheng et al./Hearing Research 105 (1997) 65-76

Fig. 5. Predominant features as seen by confocal microscopy 30 min after KA treatment, showing swollen inner radial fibers (*). Bar = 5 gm.

3.2. E V P I / 0 functions

Fig. 2 shows the mean EVP I/O functions at 1, 2, 4 and 8 kHz. Only data from ears with more than 60 dB hearing loss were included in the analysis. The I/O functions obtained before the application of K A to the round window m e m b r a n e (thin solid lines) are compared to those obtained 3 h, 3, 5 and 10 days after K A treatment. Immediately after exposure to KA, the amplitude of EVP was reduced significantly and I/O functions were depressed at all the frequencies. There was a progressive recovery in the EVP I/O functions at all the frequencies after K A application. Five to 10 days after K A treatment, the recovery had been complete and no abnormality in the EVP response could be discerned. In agreement with the changes in threshold shifts over time, recovery of EVP I/O functions at the lower frequencies (1 and 2 kHz) was delayed compared to the recovery at the higher frequencies (4 and 8 kHz). EVP I/O functions at 4 and 8 k H z had completely recovered by 5 days after K A treatment while EVP I/O functions at 1 and 2 kHz remained depressed. By Day

10, however, low-frequency EVP I/O functions had completely recovered as well. 3.3. D P O A E I/O functions

The D P O A E I/O functions at f2 frequencies of 1, 2, 4 and 8 kHz measured before, then 3 h and 5 days after K A application are presented in Fig. 3. Consistently, the mean D P O A E I/O functions were essentially unchanged relative to baseline D P O A E I/O functions at all the tested frequencies during each of the measuring sessions after K A treatment. 3.4. Histopathology

Fig. 4 demonstrates a confocal microscopic image from a control cochlea, showing a normal IHC. The pear-shaped I H C is surrounded by supporting cells. Under its basal pole, the inner spiral plexus region is packed with primary auditory afferent endings. The main histological features of KA-treated cochlea as seen with confocal microscopy 30 min after K A treat-

X.-Y. Zheng et al./Hearing Research 105 (1997) 65-76

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Fig. 6. Predominant features as seen by confocal microscopy 3 h after K A treatment. Big vacuoles formed beneath the I H C (*), by which the I H C m e m b r a n e at the basal pole was distorted (arrow). The I H C is shortened and about 1/2 of normal size. Bar = 4 gm.

ment are shown in Fig. 5. There were swollen inner radial fibers and postsynaptic afferent synapses, forming empty spaces under IHC basal pole. Three hours after K A treatment (Fig. 6), no type I afferent endings could be discerned under the IHCs and vacuoles formed beneath the IHCs as a result of massive swelling, a disruption of membranes and a loss of cytoplasmic content in the afferent dendrites. The IHC membrane at the basal pole was distorted by the vacuoles. There were no obvious pathological changes to be found under OHCs (Fig. 7). About 2 days later when the EVP threshold had recovered by 30 dB (Fig. 8), the empty space beneath the IHCs was much smaller. The basal pole of some IHCs was in normal round shape and, in some of them, surrounded by dendrites. Nerve profiles could also be seen extending toward the IHCs. N o attempt was made to quantify the dendrites. However, it is clear that the number of dendrites increased substantially between 2 and 5 days after K A treatment. Five days after exposure, hearing had returned to normal and the pattern of IHC innervation in the basal cochlea was not obviously different from normal (Fig. 9).

4. Discussion

The electrophysiological changes following K A exposure varied across earlier reports. After KA treatment, the CAP was abolished at all sound intensities (Bledsoe et al., 1981), or a significant reduction of the amplitude of CAP was primarily observable with high intensity tone burst stimulation (Puel et al., 1991a). In the current experiment, the amplitude of the EVP was reduced significantly and the I/O functions were depressed at all the tested frequencies and input levels after K A treatment. EVP I/O functions at 4 and 8 kHz were much more depressed than at lower frequencies. The discrepancy between results reported by Puel et al. (1991a) and those reported here or by Bledsoe et al. (1981) can be explained by the different routes by which the K A is given as well as the different dosages of the K A applied. When the K A is applied to the round window membrane as in the present study, it will gradually infiltrate the perilymph from the basal turn to the apical turn in the cochlea after penetrating the round

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Fig. 7. Microscopic image of the organ of Corti 3 h after KA treatment. The OHCs and its nerve endings appear normal. Bar = 5 ~tm.

window membrane. This will render a higher concentration of KA in the basal turn than in the apical one and, consequently, a stronger depressive effect of KA on EVP at the high frequencies (i.e., 4, 8 and 16 kHz) than at low frequencies (i.e., 1 and 2 kHz). It can be expected that the differences in the process of KA's penetrating the round window membrane and infiltrating the perilymph may give rise to more diversity in the KA concentration at the apex of cochlea, resulting in more fluctuations in EVP changes at apex than at base of the cochlea. This is supported by differences in variability shown in Fig. 1. However, the possibility that the basal part of the cochlea is more susceptible to the excitotoxic effect than the apex cannot be excluded (Schweitzer et al., 1991; Crofton et al., 1994). D P O A E I/O functions show that there are no significant differences in the amplitude of DPOAEs before and after KA application. The depression of EVP is consistent with the observation that KA damages IHC/VIII nerve synapses. If DPOAEs do indeed reflect O H C status as suggested (Horner et al., 1985; Schrott et al., 1989, 1991; Ohlms et al., 1991; Kujawa et al., 1992; Whitehead et al., 1992; Qiu et al., 1996; Trautwein et al., 1996), then it can be inferred that K A does not change O H C function. The possibility of having normal DPOAEs with depressed EVP function points out a possible clinical complication in interpreting DPOAEs, i.e., normal DPOAEs do not necessarily imply normal hearing.

The results of this experiment agree with the established notion that one excitotoxic effect of glutamate analogues is a massive swelling of auditory afferent dendrites under the IHCs (Pujol et al., 1985, 1993; Juiz et al., 1989; Puel et al., 1991b, 1994). However, previous studies have argued that this excitotoxic injury is irreversible (Bledsoe et al., 1981; Juiz et al., 1989). In the study of Bledsoe et al. (1981), cochlear perfusion of KA immediately abolished CAP. Artificial perilymph solution was perfused for 10 rain subsequent to the administration of KA. However, the attempt at washout produced no identical recovery of the CAP. An ultrastructural study (Juiz et al., 1989) on the longterm effect of KA on the cochlea of rat revealed a partial (34%) spiral ganglion neuron loss 10 days after treatment while swelling and rupture of inner radial fibers were still present until 30 days after the intracochlear perfusion of both artificial perilymph containing KA in experimental group and artificial perilymph alone in control group. It was concluded that the swollen fibers were non-specific in origin, presumably the result of surgical manipulations of the cochlea (Juiz et al., 1988; Rueda et al., 1989). The possibility of nonspecific effects related to cochlear perfusion has been avoided in this study by the administration of KA via the round window membrane. The additional perspective provided by the current investigation lies in two observations. First, the physiological and morphological findings in the current inves-

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Fig. 8. Predominant features as seen by confocal microscopy 2 days after K A treatment when the EVP threshold recovered by 35%. The basal part of the IHC still looks abnormal, but it is partially surrounded by neurite (arrow) and extending nerve profiles (arrowhead). Bar = 5 gm.

tigation are very consistent, clearly indicating a process of re-organization of excitotoxically damaged inner ear afferent synapses and recovery of auditory sensitivity following K A treatment. This is consistent with the study of Pujol et al. (1995) on A M P A ototoxicity. Moreover, the discrepancy between this and Bledsoe's conclusion could be explained by the timing of the recovery. Because the re-organization of auditory nerve endings and the recovery of auditory sensitivity took place in several days as shown in this investigation, it is possible that no recovery of CAP could be discerned immediately after a 10 min washout of K A from inside the cochlea in the study of Bledsoe et al. (1981). Second, we have been able to show that EVP I/O functions can fully recover after a significant depression. This gives circumstantial evidence that the repaired afferent nerve endings could render a normal sflpra-threshold auditory response in addition to a normal auditory sensitivity. These results may be relevant in terms of prevention and amelioration of some cochlear damage from ototraumatic agents (e.g., intense loud noise, is-

chemia and ototoxic drugs) that may cause massive swelling and destruction of postsynaptic endings of the auditory nerve (Spoendlin, 1971; Robertson, 1983; Pujol et al., 1993). One thing should be noted that the complete recovery of auditory supra-threshold response as reported here may imply that the entire population of the auditory nerve fibers are functioning normally. This is in contradictory to the study of Juiz et al. (1989) that there was a partial (1/3)ganglion neuron loss after K A treatment. The current data obtained by measuring EVP from the inferior colliculus does not resolve this discrepancy because there is a possibility of central plasticity involved in the normalization of the EVP I/O function after K A ototoxicity. Further studies using CAP and auditory nerve single unit responses as indices are necessary. Our current data do not allow a d|rect statement about the nature of the VIII nerve afferent damage, i.e., is it regeneration? However, the present study raises the issue of regeneration and neo-synaptogenesis

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Fig. 9. Predominant features as seen by confocal microscopy 5 days after KA treatment. No swelling of the afferent dendrites under the IHC could be discerned. Under its basal pole, the inner spiral plexus region is packed with nerve endings. Bar = 5 gm.

of auditory nerve dendrites after excitotoxic injuries as claimed by Pujol et al. (1995) and Puel et al. (1995). Although we lack direct evidence of the mechanisms involved in this process, it is nevertheless interesting to speculate about possible contributors. One possibility is that the olivocochlear (OC) efferent system may play a role. The following observations would suggest such a role. First, neurotransmitters in the lateral OC fibers projecting to the afferent terminals in the region under the I H C s might act as modulators promoting the subsidence of the swelling and the recovery of the function. A m o n g the neurotransmitters localized to the lateral OC fibers, dopamine and calcium-gene-related peptide have been demonstrated to have a protective or recuperative role (Pujol et al., 1993; Pujol, 1994). Second, a trophic action has previously been proposed for the lateral OC system (Liberman, 1990; Pujol, 1994). Third, the activities of cholinergic metabolism increase form apex to base (Godfrey et al., 1976; Godfrey and Ross, 1985), which could conceivably account for the base versus apex difference in rate of recovery.

5. Conclusion The present study demonstrates that application of K A to the round window membrane of chinchilla causes immediate swelling and disruption of IHC/afferent dendrites of primary auditory neurons, and a significant reduction of EVP amplitudes. By contrast, D P O A E I/O functions remained unchanged, indicating that O H C s were not affected by K A application and that intact I H C N I I I nerve synapses are not needed for normal DPOAEs. W h a t is more interesting is that the nerve ending swellings have subsided and the damaged nerve endings have recovered, rendering a normal auditory sensitivity and supra-threshold response as revealed by EVP I/O functions within 5 10 days after excitotoxic injury.

Acknowledgments This work was supported by grants from N I H 1R01

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DC1237-01A1. The authors wish to thank Drs. Richard J. Salvi and Robert Burkard for critical comments on the manuscript and Nicholas Powers for his expert technical assistance in data collection. References Altschuler, R.A., Sheridan, C.E., Horn, J.W. and Wenthold, R.J. (1989) Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea. Hear. Res. 42, 167 173. Bergman, B.M., Gorga, M.P., Neely, S.T., Kaminski, J.R., Beauchaine, K.L. and Peters, J. (1995) Preliminary descriptions of transient-evoked and distortion-product otoacoustic emissions from graduates of an intensive care nursery. J. Am. Acad. Audiol. 6, 150~162. Bledsoe, S.C., Jr., Bobbin, R.P. and Chihal, D.M. (1981) Kainic acid: an evaluation of its action on cochlear potentials. Hear. Res. 4, 109 120. Coyle, J.T. (1983) Neurotoxic action of kainic acid. J. Neurochem. 41, 1-11. Crofton, K.M., Janssen, R., Prazma, J., Pulver, S. and Barone, S. (1994) The totoxicity of 3,3'-iminodipropionitrile: functional and morphological evidence of cochlear damage. Hear. Res. 80, 129 140. Dolan, D.F., Nuttall, A.L. and Avinash, G. (1990) Asynchronous neural activity recorded from the round window. J. Acoust. Soc. Am. 87, 2621-2627. Felix, D. and Ehrenberger, K. (1990) A microiontophoretic study of the role of excitatory amino acids at the afferent synapses of mammalian inner hair cells. Eur. Arch. Oto-Rhino-Laryngol. 248, 1 3. Godfrey, D.A. and Ross, C.D. (1985) Enzymes of acetylcholine metabolism in the rat cochlea. Ann. Otol. Rhinol. Laryngol. 94, 409414. Godfrey, D.A., Krzanowski, J.J. and Matschinsky, F.M. (1976) Activities of enzymes of the cholinergic system in the guinea pig cochlea. J. Histochem. Cytochem. 24, 470-472. Hall, J.W., III, Baer, J.E., Chase, P.A. and Schwaber, M.K. (1994) Clinical application of otoacoustic emissions: what do we know about factors influencing measurement and analysis? [Review]. Otolaryngol.-Head Neck Surg. 110, 22 38. Henderson, D., Hamernik, R.P., Woodford, C., Sitler, R.W. and Salvi, R.J. (1973) Evoked response audibility curve of the chinchilla. J. Acoust. Soc. Am. 54, 1099-1101. Hornet, K.C., Lenoir, M. and Bock, G.R. (1985) Distortion product otoacoustic emissions in hearing impaired mice. J. Acoust. Soc. Am. 78, 1603 1611. Janssen, R., Schweitzer, L. and Jensen, K.F. (1991) Glutamate neurotoxicity in the developing rat cochlea: physiological and morphological approaches, Brain Res. 552, 255-264. Juiz, J.M., Rueda, J. and Merchan, J.A. (1988) Reversible damage to the nerve fibres in the organ of Corti after surgical opening of the cochlea in the rat. Acta Oto-Laryngol. 106, 29-33. Juiz, J.M., Rueda, J., Merchan, J.A. and Sala, M.L. (1989) The effects of kainic acid on the cochlear ganglion of the rat. Hear. Res. 40, 65-74. Kujawa, S.G., Glattke, T.J., Fallon, M. and Bobbin, R.P. (1992) Intracochlear application of acetylcholine alters sound-induced mechanical events within the cochlear partition. Hear. Res. 61, 106-116. Lefebvre, P.P., Weber, T., Leprince, P., Rigo, J.M., Delree, P., Rogister, B. and Moonen, G. (1991) Kainate and N M D A toxicity for cultured developing and adult rat spiral ganglion neurons: further evidence for a glutamatergic excitatory neurotransInission at the inner hair cell synapse. Brain Res. 555, 75-83.

75

Li, H.S., Niedzielski, A.S., Beisel, K.W., Hiel, H., Wenthold, R.J. and Morley, B.J. (1994) Identification of a glutamate/aspartate transporter in the rat cochlea. Hear. Res. 78, 235542. Liberman, M.C. (1990) Effects of chronic cochlear de-efferentation on auditory-nerve response. Hear. Res. 49, 209 224. Lonsbury-Martin, B.L. and Martin, G.K. (1990) The clinical utility of distortion-product otoacoustic emissions. [Review]. Ear Hear. 11, 144~154. Nakagawa, T., Komune, S., Uemura, T. and Akaike, N. (1991) Excitatory amino acid response in isolated spiral ganglion cells of guinea pig cochlea. J. Neurophysiol. 65, 715-723. Ohlms, L.A., Lonsbury-Martin, B.L. and Martin, G.K. (1991) Acoustic-distortion products: separation of sensory from neural dysfunction in sensorineural hearing loss in human beings and rabbits. Otolaryngol.-Head Neck Surg. 104, 159 174. Puel, J.L., Ladrech, S., Chabert, R., Pujol, R. and Eybalin, M. (1991a) Electrophysiological evidence for the presence of NMDA receptors in the guinea pig cochlea. Hear. Res. 51, 255-264. Puel, J.L., Pujol, R., Ladrech, S. and Eybalin, M. (1991b) Alphaamino-3-hydroxy-5-methyl-4-isoxazole propionic acid electrophysiological and neurotoxic effects in the guinea-pig cochlea. Neuroscience 45, 63-72. Puel, J.L., Pujol, R., Tribillac, F., Ladrech, S. and Eybalin, M. (1994) Excitatory amino acid antagonists protect cochlear auditory neurons from excitotoxicity. J. Comp. Neurol. 341, 241-256. Puel, J.L., Saffiedine, S., Gervais d'Aldin, C., Eybalin, M. and Pujol, R. (1995) Synaptic regeneration and functional recovery after excitotoxic injury in the guinea pig cochlea. Comptes Rendus de l'Academie des S c i e n c e s - Serie Iii, Sciences de la Vie. 318, 67 75. Pujol, R. (1994) Lateral and medial efferents: a double neurochemical mechanism to protect and regulate inner and outer hair cell function in the cochlea. Br. J. Audiol. 28, 185-191. 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. Pujol, R., Puel, J.L., Gervais d'Aldin, C. and Eybalin, M. (1993) Pathophysiology of the glutamatergic synapses in the cochlea. Acta Oto-Laryngol. 113, 330-334. Pujol, R., Gervais d'Aldin, C., Saffiedine, S., Eybalin, M. and Puel, J.L. (1995) Repair of inner hair cell-auditory nerve synapses and recovery of function after an excitotoxic injury. In: R.J. Salvi, D. Henderson, F. Fiorino and V. Colletti (Eds.), Auditory System Plasticity and Regeneration, Thieme Press, New York, pp. 1 0 ~ 107. Qiu, C.X., Salvi, R.J., Hofstetter, P. and Powers, N.L. (1996) Selective IHC loss significantly reduces the neural output of the cochlea, but does not alter threshold or the evoked response amplitude of the auditory cortex. ARO Midwinter Meeting XIX, p. 112. Robertson, D. (1983) Functional significance of dendritic swelling after loud sounds in the guinea pig cochlea. Hear. Res. 9, 263-278. Rothman, S.M. and Olney, J.W. (1987) Excitotoxicity and the NMDA receptor. Trends Neurosci. 10, 29~302. Rueda, J., Juiz, J.M. and Merchan, J.A. (1989) Surgical trauma to the cochlea results in reversible damage to spiral ganglion type I neurons. Acta Oto-Laryngol. 107, 59-62. Schron, A., Stephan, K. and Spoendlin, H. (1989) Hearing with selective inner hair cell loss. Hear. Res. 40, 213-220. Schrott, A., Puel, J.-L. and G. Rebillard (1991) Cochlear origin o f 2fl-f2 distortion products assessed by using two types of mutant mice. Hear. Res. 52, 245-254. Schweitzer, L., Jensen, K.F. and Janssen, R. (1991) Glutamate neurotoxicity in rat auditory system: cochlear nuclear complex. Neurotoxicol. Teratol. 13, 189 193. Spoendlin, H. (1971) Primary structural changes in the organ of Corti after acoustic overstimulation. Acta Oto-Laryngol. 71, 166-176. Subramaniam, M., Salvi, R.J., Spongr, V.P., Henderson, D. and Powers, N.L. (1994) Changes in distortion product otoacoustic

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emissions and outer hair cells following interrupted noise exposures. Hear. Res. 74, 204-216. Trautwein, P., Hofstetter, P., Wang, J. and Salvi, J. (1996) Selective inner hair cell loss does not alter distortion product otoacoustic emissions. Abstr. Assoc. Res. Ototaryngol., p. 94. Whitehead, M.L., Lonsbury-Martin, B.L. and Martin, G.K. (1992)

Evidence for two discrete sources of 2fl f2 distortion-product otoacoustic emission in rabbit. II. Differential physiological vulnerability. J. Acoust. Soc. Am. 92, 2662-2682. Zheng, X.Y., Wang, J., Salvi, R.J. and Henderson, D. (1996) Effects of kainic acid on the cochlear potentials and distortion product otoacoustic emissions in chinchilla. Hear. Res. 95, 161 167.