EXPERIMENTAL
NEUROLOGY
46, 542-553
(1975)
Noise-Induced Hearing Loss, Auditory Evoked Potentials, and Protection from Audiogenic Seizures in Mice JAMES F. WILLOTT, Department
KENNETH
R. HENRY, AND FRANK GEORGE l
of Psychology, University DazEis, California 95616 Received
October
of California,
13, 1974
Protection from audiogenic seizures by exposing susceptible mice to intense acoustic stimulation prior to seizure testing was investigated in three experiments. Auditory evoked potentials were measured from the inferior colliculi of two strains of mouse, the C57BL/6J and DBA/ZJ, and were related to parallel measures of the acoustic startle (Preyer) response and audiogenie seizures. Exposed groups were subjected to intense sound while they were anesthetized with ether prior to testing. Control groups were treated identically but not exposed to sound before testing. As a result of this prior exposure to sound, audiogenic seizures were reduced, Preyer thresholds were elevated, and the absolute threshold for auditory evoked potentials was elevated. However, at intensities sufficient to induce audiogenic seizures, the amplitudes of the auditory evoked potentials were not affected. These results suggest that whatever noise-induced hearing loss occurred, it was not observed at high intensities. Interference with patterns of neural activity was suggested as the mechanism of protection from seizures.
INTRODUCTION Strains of mice such as the C57BL/6J are normally not susceptible to audiogenic seizures, yet they may be rendered susceptible by briefly exposing them to intense sound and allowing about 5 days to pass (acoustic priming ; 4). A recent study (8) showed that such primed mice were protected from sound-induced convulsions by exposing them to a secondintense sound (electric bell while under light ether anesthesia) 4 hr before testing for seizures. Since this protection from audiogenic seizures was accompanied by an elevation of the Preyer (acoustic startle response) threshold, it was suggested that reduction of seizures was simply the result of noise-induced hearing loss: i.e., the magnitude of neural activity necessary 1 Supported K.R.H.
by NSF Grant No. GB 31921 and PHS
542 Copyright All rights
s
1975 by Academic Press, Inc. reproduction in any form reserved.
Grant No. NS 11565-01 to
AUDITORY
543
FUNCTION
to initiate audiogenic seizures, which is normally evoked by an electric bell, was not realized because of impaired auditory function, so that the animals were protected from seizures. The experiments reported below were designed to test this hypothesis with the use of auditory evoked potentials, the best method available to examine noise-induced hearing loss in laboratory mice. Hearing loss should be reflected in the auditory evoked potential by elevated absolute thresholds and depression of suprathreshold amplitudes (2, 3, 12, 13). Experiment 1 was a preliminary experiment in which our previous (8) study was replicated in every detail, except that instead of observing Preyer responses and audiogenic seizures, auditory evoked potentials were recorded. Experiments 2 and 3 used a different strain of mouse and were designed to provide a more controlled examination of the relationship between audiogenic seizures and noise-induced hearing loss as measured by Preyer thresholds, and thresholds and amplitudes of auditory evoked potentials. EXPERIMENT
1
Method Twenty C57BL/6 J mice were acoustically primed at age 16 days and tested at age 21 days, the 5 day lapse being optimum for the development of susceptibility to audiogenic seizures (4). On the day of testing, all subjects were anesthetized lightly with ether. Ten subjects (exposed group) were exposed to the sound of a 5 cm electric bell (mean intensity 114 db SPL) in a glass bell jar for 30 sec. Etherization was necessary to prevent seizures during this exposure to sound. The remaining ten subjects (control group) were also etherized but were not exposed to sound. Four hours later, all subjects were anesthetized with sodium pentobarbital (Nembutal 1.5 pg/g of a 50 mg/db solution) for recording of auditory evoked potentials. This was the first departure from our previous experiment in which subjects were tested for Preyer thresholds and audiogenic seizures instead of being anesthetized, In line with our previous data, auditory evoked potentials of the exposed group should be associated with a state of low susceptibility to seizures, whereas auditory evoked potentials from the control group would be those obtained from highly susceptible animals. The method for obtaining auditory evoked potentials has been described in detail (18). Briefly, anesthetized subjects were placed in a stereotaxic headholder which did not utilize earbars. The skull was exposed, and bipolar steel electrodes were lowered through the soft skull into the inferior colliculus. The contralateral ear was presented with a lOkHz-20kHz noise band (25 msec bursts, 1 msec rise and decay, 4 pulses/set) which
544
WILLOTT,
HENRY
AND GEORGE
was controlled without producing transients. Sound was delivered through a T-shaped metal tube, with one opening going to the subject’s ear and the other to the microphone of a Bruel and Kjoer SPL meter. This provided a close approximation of the sound pressure at the subject’s ear. Auditory evoked potentials were amplified and relayed to an oscilloscope. The subject’s absolute threshold for auditory evoked potentials was initially measured. This parameter, defined as the lowest intensity which evoked a visible (2 pv) response on the oscilloscope, provided an estimate of absolute auditory sensitivity. Then the intensity of the acoustic stimulus was increased in 10 db steps to 100 db, the maximum intensity our system could deliver without disortion. At each 10 db increment, the peak-topeak auditory evoked potentials of the amplitude was recorded. RESULTS Figure 1 depicts input-output functions for the exposed and control groups. The absolute auditory evoked potential threshold was elevated by 5.8 db in the exposed group [t( 18) = 2.47, P < 0.051, undoubtedly reflecting the threshold shift which typically accompanies exposure to intense sounds. At moderate intensities, there appears to be some depression of the auditory evoked potential amplitude in the exposed curve, but the greatest differences are not quite statistically significant with a one-tailed t-test. It is quite evident that at the highest intensities there was no difference in auditory evoked potential amplitude. The results of this preliminary experiment are clear-cut. While there was an elevation of the absolute auditory evoked potential threshold,
FIG. 1. Input-output
functions for C57BL/6J
mice of Expt. 1.
AUDITORY
545
FUNCTION
suprathreshold amplitudes showed little if any effects of exposure to the intense noise, despite the fact that such exposure reduces audiogenic seizures and elevates Preyer thresholds. In order to adequately interpret these results, however, it was necessary to provide greater stimulus control for two reasons. First, in Expt 1 an electric bell was used for noise exposure, while a 10-20 kHz noise band was used for auditory evoked potentials. This noise band includes the frequencies most effective in inducing audiogenie seizures (7) but the bell could have affected responses to very high or low frequencies which could conceivably influence seizures. Such effects could have gone undetected by auditory evoked potentials evoked by the noise band. Second, the maximum intensity we were able to provide for auditory evoked potentials was 100 db SPL, whereas the electric bell provided a mean intensity of 114 db. Furthermore, the aperiodicity and irregular energy distribution of the electric bell could be important factors. It was therefore necessary to perform the following experiments in which these variables were more tightly controlled. EXPERIMENT
2
Method This experiment was designed to minimize differences in the acoustic stimuli used for noise exposure, for inducing seizures, for measuring Preyer thresholds, and for obtaining auditory evoked potentials. Also, DBA/2J mice were used as subjects to eliminate the need for priming, as well as to extend our research to another strain of mouse. This strain is innately highly susceptible to audiogenic seizures, allowing a less intense sound to be used to induce seizures. New and improved equipment also aided the present and following experiments. Thirty-nine 23-24 day-old DBA/2 J mice served as subjects. On the day of testing, all subjects were lightly anesthetized with ether, again, to protect those which were exposed to noise from having seizures. The exposed group was placed in a 20 X 15 cm diameter plexiglass cylinder placed upright on the floor of a sound proofed box. At the top of the cylinder an Altec 802 tweeter housed in a 17 X 19 X 19 cm box provided the acoustic stimulus, which was 90 sec. duration of a 10-20 kHz noise band at a mean intensity of 120 db SPL (sound pressure level). This was ascertained by initially measuring at the position of the mouse in 1 kHz bands. The SPL was 130 db at 10 kHz and dropped linearly to 110 db at 20 kHz. This closely approximated the spectral and intensity characteristics measured at the ear of the mouse when auditory evoked potentials were obtained in Expt 3. The control group was treated identically, but not exposed to the noise. Fifteen to 20 min later, after recovery from the effects of ether,
546
WILLOTT,
HENRY
AND
GEORGE
Preyer thresholds were obtained and subjects were tested for audiogenic seizures. For Preyer thresholds, either control (ten) or exposed (ten) subjects were replaced into the sound chamber. Preyer responses were elicited by brief bursts of the same 10-20 kHz noise band (25 msec rise and decay time). Preyer threshold was obtained by varying the intensity of the noise bursts until the minimum level capable of eliciting the startle response was found. For each of the above subjects, immediately after the Preyer threshold was obtained, the intensity of the noise band was raised to 110 db SPL for 1 min to test for audiogenic seizures. An additional 19 subjects had been previously tested for seizures in a pilot study and their data are included here. For ‘every subject, the occurrence of each scccessive stage of audiogenie seizures-wild running, clonic seizure, tonic seizure, and death-was recorded, and latency to wild running was measured with a stopwatch. Results The results of Expt. 2 are presented in Table 1. The data clearly show an elevation of the Preyer threshold for the exposed group. This group had a mean Preyer threshold of 102 db SPL compared to 87 db for the control group [ t( 18) = 7.49, P < O.OOl] . The elevation of the Preyer threshold was comparable to that obtained from C57BL/6J mice in the previously cited study (8). Although that study used an electric bell for noise exposure, it used the same lo-20 kH z noise band for Preyer responses. Table 1 also shows that the exposed subjects were clearly protected from audiogenic seizures. For the exposed group, the mean latency to seizures (wild running stage) was increased by 3.1 sec. [ t(38) = 4.331, TABLE PREYER
THRESHOLDS
AND SEVERITY
1 OF AUDIOGENIC
SEIZURES
(EXPT
2)a
Parameter
Control
Exposed
Preyer threshold Mean latency to wild running Mean severity of seizureb Incidence of seizures Incidence of deaths
87 db 2.9 set 3.6 19 of 19 subjects 15 of 19 subjects
101 db 5.0 set 1.2 11 of 20 subjects 4 of 20 subjects
a Subjects were DBA/2J mice. The exposed group was subjected, thesia, to 90 set of a lo-20 kHz noise band, 15-20 min before data co&o2 group was treated identically, but not exposed to sound. b Severity was scored as: 1 = wild running; 2 = clonic seizure; 4 = death (5).
under ether was obtained. 3 = tonic
anesThe seizure;
AUDITORY
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FUNCTION
P < O.OOl], and the mean severity of seizures was decreased to one-third that of controls [t(38) = 6.082, P < O.OOl]. Also, the incidence of seizures for the control group was 100% compared to 55% for exposed (x” = 8.725, P < O.OOl), and the incidence of lethal seizures was 79% for controIs compared to 20% for exposed subjects (x2 = 11,295, P < 0.001). EXPERIMENT
3
Method The final experiment was a replication of Expt. 2, except that instead of observing Preyer responses and audiogenic seizures, auditory evoked potentials were recorded. This allowed a determination of noise-induced hearing loss, as measured by auditory evoked potential parameters, which accompanied the elevated Preyer thresholds and reduced audiogenic seizures observed in Expt. 2. The main improvement in this experiment is that the same noise band was used for auditory evoked potentials as was used for acoustic exposure, Preyer responses, and audiogenic seizures. This markedly reduced the possibility of extraneous effects of noise exposure on the auditory system which might not be detected by auditory evoked potentials-a deficiency of Expt. 1. Twenty-six DBA/2J mice served as subjects. Exposed and control groups were treated the same as their counterparts in Expt. 2, except that auditory evoked potentials were recorded rather than measurement of Preyer thresholds and audiogenic seizures. The apparatus used to obtain auditory evoked potentials was similar to that described in Experiment 1, with several modifications. Most importantly, an Altec 802 tweeter replaced the Mustang Spheracon tweeter to provide the acoustic stimulus, allowing an intensity of 110 db to be delivered to the mouse’s ear in the stereotaxic apparatus. Thus, auditory evoked potentials in response to the intensity used to induce seizures could be recorded. Also, on-line computer averaging was obtained from ten mice with an Ortec signal averaging system, permitting more detailed analysis of auditory evoked potential waveform. The procedure for obtaining AEPs was also modified. Rather than obtaining full input-output functions, only absolute AEP thresholds and 110 db auditory evoked potentials were recorded. This reduced the possibility of auditory fatigue during auditory evoked potential measurement and limited suprathreshold measurement to the intensity used to induce seizures. The auditory evoked potential at this latter intensity should be most relevant to audiogenic seizures. An additional procedure was also added to examine possible differences in “fatigability” of the ears of exposed and control groups. Since seizures
548
WILLOTT,
HENRY
AND
GEORGE
begin after a latency of several seconds and their time course may run from 30 to 60 set, it is important to know if auditory fatigue during seizures could lead to reduced magnitude of neural activity. That is, did the exposed animals have increased vulnerability to auditory fatigue which reduced the severity of seizures by decreasing neural input during the initial stages of seizures? To test this possibility, subjects were exposed to two additional 15 set periods of noise while in the stereotaxic apparatus. After the initial auditory evoked potential threshold and 110 db auditory evoked potential had been recorded, 15 set of the noise band at 110 db were given. The auditory evoked potential was recorded immediately after. Next, a second additional 15 set noise exposure was given, and another auditory evoked potential was then recorded. Thus, for both exposed and control groups, the following auditory evoked potential parameters were obtained : (a) an initial absolute auditory evoked potential threshold ; (b) an initial auditory evoked potential in response to 110 db ; (c) a second auditory evoked potential in responseto 110 db, but following an additional 15 set noise exposure ; and (d) a third 110 db following still another 15 set noise exposure. RESULTS For the exposed group the absolute auditory evoked potential threshold was 42.1 db compared to 23.2 db for the control group [t (24) = 3.447, P < 0.011, as seen in Tbble 2. This is consistent with the results of Expt. 1 and is indicative of the expected noise-induced threshold shift. However, the peak-to-peak amplitude of the initial auditory evoked potential at 110 db was not affected, as can be seen in Fig. 2. Furthermore, latency and waveform did not differ between the two groups. These results are clearly in accord with those of Expt. 1: whereas noise exposure was followed by TABLE AUDITORY
AEP AEP AEP AEP
threshold amplitude amp. after amp. after
EVOKED
POTENTIAL
2 PARAMETERS
FOR EXPT
Parameter
Control
(110 db) first additional exposure second additional exposure
23.2 db 74 flv 62 pv 58 /N
a Subjects were DBA/ZJ mice which had either been exposed of 110 db of a lo-20 kHz noise band 15-20 min before auditory ing. “Additional exposures” were 1.5 set of the same noise band were in the stereotaxis apparatus. ) P(C = E) < 0.01.
3a Exposed 42.1 db” 83 /N 65 /N 51 )bv
or not exposed evoked potential at 110 db while
to 90 set recordsubjects
AUDITORY
FIG. 2. Peak-to-peak
549
FUNCTION
AEP amplitude for DBA/ZJ
mice of Expt. 3.
an increase in absolute auditory evoked potential threshold, high intensity auditory evoked potential amplitudes were not affected. The results of the second and third 15 set noise exposure are also seen in Fig. 2. For both control and exposed groups, the two additional noise exposures were effective in reducing auditory evoked potential amplitude immediately after exposure. Analysis of variance for repeated measures showed both groups had significant reductions in auditory evoked POtential amplitudes [exposed: F (2.24 = 22.598, P < 0.001; control: F(2.24) = 6.225, P < 0.011 as a result of the additional exposures. Further analysis indicates that the greatest reduction of auditory evoked potential amplitude occurred following the first additional exposure for both control [t(l2) = 2.355, P < 0.05] and en-fiosed [t(12) = 4.534, P < 0.0005]. Although it appears from Fig. 2 that the curve for the exposed group has a steeper decline than that for the control group, the curves did not differ significantly as indicated by the lack of interaction between conditions (control, exposed) and additional exposures [F (2.48) = 2.279, P > 0.11. It appears from these data that the additional noise exposures were effective in reducing the amplitude of the 110 db auditory evoked potential, but there was little if any difference in the magnitude of this effect between control and exposed groups. Using this as our criterion, it appears that the exposed animals were not significantly more susceptible to auditory fatigue than the controls. DISCUSSION The present study set out to determine whether or not protection from audiogenic seizures could be accounted for by noise-induced hearing loss. The data indicate that the answer is not a simple one. The elevated
550
WILLOTT,
HENRY
AND
GEORGE
Preyer thresholds and auditory evoked potential thresholds indicate that hearing loss did occur, whereas the auditory evoked potential amplitudes at seizure-inducing intensity provides no evidence of hearing loss. It is pertinent to first examine the nature of noise-induced hearing loss in terms of the auditory evoked potential threshold, Preyer threshold, and auditory evoked potential amplitude and then discuss their relevance to audiogenic seizures. The elevation of absolute auditory evoked potential threshold indicates that hearing loss was evident at low intensities. Such threshold shifts have been demonstrated at various levels of the auditory system following exposure to noise (2, 3, 12, 13, 17, 18). Furthermore, elevation of the thresholds for auditory evoked potentials has been directly associated with behaviorally measured hearing loss (12). Elevation of the Preyer threshold has also been reliably associated with exposure to intense noise and with conductive hearing loss (8, and an unpublished study in our laboratory). It seems quite certain that the consistent elevation of the Preyer threshold following noise exposure in the present study is a function of hearing loss at moderate intensity. The amplitude of the auditory evoked potential at high intensities of stimulation has also been shown to be depressed by exposure to intense sound. This has been demonstrated in both the peripheral and central auditory pathway (2, 12, 13, 17, 18). In fact, Expt. 3 showed that exposure to additional periods of noise resulted in depression of the 110 db auditory evoked potentials immediately after exposure (Fig. 2). Depression of the auditory evoked potential amplitude, therefore, seems to be a reliable indicator of hearing loss at higher intensities. The lack of effect on the auditory evoked potentials of the exposed groups of Expt. 1 and 3 therefore indicates that the subjects rapidly recovered from whatever high intensity hearing loss they may have suffered. If then, the auditory evoked potential threshold, Preyer threshold, and auditory evoked potential amplitude are all reliable indices of hearing loss, one must conclude that hearing loss was nonlinear in the present study. That is, there was a loss at low and moderate intensities auditory evoked potential and Preyer threshold elevations) but no diminution of high intensity responses (auditory evoked potential amplitudes). This type of relationship has in fact been reported for both animal auditory evoked potential and human loudness experiments. In animals, Rosenblith et al. (13) found that the effects of high intensity sound exposure were greater for low intensity than for high intensity responses, as indicated by the cortical auditory evoked potential of cats. Recently, Pugh et al. (12) showed that, while the suprathreshold eighth nerve potential was initially depressed by sound exposure, its rate of growth increased rapidly despite
AUDITORY
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threshold elevation. Similar effects have been demonstrated with auditory evoked potential in mice (6, 14, 17, 18). With human subjects, Davis et al. (1) showed that noise exposure elevated absolute thresholds of hearing but had little effect on the loudness of higher intensity sound. A similar effect was reported by Small and Canakl (15), who found that temporary threshold shifts were much greater than loudness shifts following sound exposure. McPherson et al. (11) observed that loudness and threshold shifts may recover at different rates. In light of such evidence, it seems quite reasonable that the high intensity responsiveness of the exposed groups recovered by the time of testing for audiogenic seizures. The slower recovering threshold and Preyer responses,however, still indicated hearing loss. If auditory evoked potential amplitudes at seizure-inducing intensities are not depressed by the noise exposures used in the present study, what is the mechanism of protection from seizures? Clearly, a general reduction of the magnitude of neural activity evoked by sound, as measured by the auditory evoked potential, cannot account for the inhibition of seizures. Such a reduction of seizures should be associated with decreased auditory evoked potential amplitude. It is more likely that the mechanism of protection from audiogenic seizures may instead involve disruption of patterns of neural activity normally associated with seizures. It is most certain that different populations of receptors and neurons are functional before and after exposure to noise. Some components, those most vulnerable to trauma, are eliminated by auditory fatigue or damage. The loss of such responsesare apparently most evident in such measuresas absolute threshold and Preyer threshold. In light of the present findings, it seems that those elements of the auditory system which have been so affected are necessary for the initiation of audiogenic seizures. What processes are involved are at present unknown, but it does seemthat the specific pattern rather than magnitude of neural activity is most important in protection from seizures. This interpretation is consistent with a previous study from our laboratory which demonstrated protection from seizures by physical restraint of movement ( 15), where it seemedmost likely that disruption of normal patterns of neural activity was involved. With regard to the relationship between auditory evoked potential amplitude of the susceptibility to audiogenic seizures, the results of the present investigation provide evidence that the two are not closely related. Highly susceptible (control) mice and low susceptible (exposed) mice had auditory evoked potential amplitudes which did not differ. These results are in agreement with several recent reports, which also failed to find this relationship (9, 10, 18). Such findings do not, however, support earlier hypotheses which associatedacoustic priming for audiogenic seizures
552
WILLOTT,
HENRY
AND
GEORGE
with “disuse supersensitivity” (6, 14). These studies demonstrated that, following priming, auditory evoked potential amplitudes became elevated as susceptibility to audiogenic seizures developed. It was hypothesized that the high auditory evoked potential amplitude indicated supersensitivity of the nervous system which resulted in susceptibility to audiogenic seizures. The present study and the other studies cited indicate that, whatever might be the cause of auditory evoked potential amplitude increased following priming, it is not responsible for audiogenic seizures. It is evident that an explanation of audiogenic seizures must go further than simply considering the amplitude of neural activity. In summary, the data reported here demonstrate that the effects of exposing the auditory system to noise are more complex than one might suppose, at least in regard to the inhibition of audiogenic seizures. The results of two different experimental designs using two strains of mouse suggest that specific patterns of neural activity in the auditory system are crucial in the initiation of audiogenic seizures. Exposure to noise may interfere with the functioning of elements in the auditory system which are most vulnerable to damage and thereby disrupt patterns of neural activity which normally are necessary for seizures. The fact that disruption of these patterns results in protection from audiogenic seizures may carry some implication for protection from other types of seizures. REFERENCES H., C. T. MORGAN, J. E. HAWKINS, R. GALAMBOS, and F. W. SMITH. 1950. Temporary deafness following exposure to loud tones and noise. Acta
1. DAVIS,
oto-Luryngol. 2. DAVIS, H., 3. 4. 5.
6.
suppz.
88.
et al. 1953. Acoustic trauma in the guinea pig. J. Acoust. Sot. Amer. 25 : 1180-1189. GISSELSSON, L., and H. SORENSEN. 1960. Auditory adaptation and fatigue in cochlear potentials. Actu Oto-Laryngol. 50: 391-405. HENRY, K. R. 1967. Audiogenic seizure susceptibility induced in C57B1/6J mice by prior auditory exposure. Science 158: 938-940. HENRY, K. R., and R. E. BOWMAN. 1969. Effects of acoustic priming on audioand chemoconvulsive seizures. J. Camp. Physiol. genie, electroconvulsive, Psychol. 67 : 401-406. HENRY, K. R., and M. SALEH. 1973. Recruitment deafness: functional effect of priming induced audiogenic seizures in mice. J. Comb. Physiol. Psychol. 84:
430-43s. 7. HENRY, K.
teristics
R., K. A. THOMPSON, and R. F. BOWMAN. 1971. Frequency characof acoustic priming by audiogenic seizures in mice. Exp. Neural.
31: 402-407. 8. HENRY, K. R.,
and J. F. WILLOTT. 1972. Unilateral inhabition of audiogenic seizures and Preyer reflexes. Nature (London) 240 : 481-482. 9. MCGINN, M. D., and K. R. HENRY. Acute versus chronic acoustic deprivation: Effects of auditory evoked potentials and seizures. Dev. Psychobiol. (in press).
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10. MCGINN, M. D., J. F. WILLOTT, and K. R. HENRY. 1973. Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature [New Biol.] 244: 255-256. 11. MCPHERSON, D., D. FRANK, and C. V. ANDERSON. 1970. Relation of temporary loudness shift to temporary threshold shift. J. Acoust. Sot. Amer. 49: 11951202. 12. PUGH, J. E., M. R. MILTON, and D. J. ANDERSON. 1974. Cochlear electrical activity in noise-induced hearing loss. Arch. Otolar. 100 : 36-40. 13. ROSENBLITH, W. A., R. GALAMBOS, and I. HIRSH. 1950. The effect of exposure to loud tones upon animal and human responses to acoustic clicks. Scieme 111: 569-571. 14. SAUNDERS, J. C., G. R. BOCK, R. JAMES, and C. S. CHEN. 197.2. Effects of priming for audiogenic seizures on auditory evoked responses in the cochlear nucleus and inferior colliculus of BALB/c mice. ExD. Neural. 37: 38-394. 15. SMALL, A., and J. A. CANAHL. 1965. Loudness changes in a forward-masking stimulus paradigm. J. Acoust. Sot. Amer. 38: 928. 16. WILLOTT, J. F. 1974. Protection from lethal audiogenic seizures in mice by physical restraint of movement. Exp. Neural. 43: 359-368. 17. WILLOTT, J. F. Some effects of exposure to intense acoustic stimulation on input-output functions in mice. J. Aud. Rex. (in press). 18. WILLOTT, J. F., and K. R. HENRY. 1974. Auditory evoked potentials: developmental changes of threshold and amplitude following early acoustic trauma. J. Camp. Physiol. Psychol. 86: 1-7.
NEUROLOGY
46, 542-553
(1975)
Noise-Induced Hearing Loss, Auditory Evoked Potentials, and Protection from Audiogenic Seizures in Mice JAMES F. WILLOTT, Department
KENNETH
R. HENRY, AND FRANK GEORGE l
of Psychology, University DazEis, California 95616 Received
October
of California,
13, 1974
Protection from audiogenic seizures by exposing susceptible mice to intense acoustic stimulation prior to seizure testing was investigated in three experiments. Auditory evoked potentials were measured from the inferior colliculi of two strains of mouse, the C57BL/6J and DBA/ZJ, and were related to parallel measures of the acoustic startle (Preyer) response and audiogenie seizures. Exposed groups were subjected to intense sound while they were anesthetized with ether prior to testing. Control groups were treated identically but not exposed to sound before testing. As a result of this prior exposure to sound, audiogenic seizures were reduced, Preyer thresholds were elevated, and the absolute threshold for auditory evoked potentials was elevated. However, at intensities sufficient to induce audiogenic seizures, the amplitudes of the auditory evoked potentials were not affected. These results suggest that whatever noise-induced hearing loss occurred, it was not observed at high intensities. Interference with patterns of neural activity was suggested as the mechanism of protection from seizures.
INTRODUCTION Strains of mice such as the C57BL/6J are normally not susceptible to audiogenic seizures, yet they may be rendered susceptible by briefly exposing them to intense sound and allowing about 5 days to pass (acoustic priming ; 4). A recent study (8) showed that such primed mice were protected from sound-induced convulsions by exposing them to a secondintense sound (electric bell while under light ether anesthesia) 4 hr before testing for seizures. Since this protection from audiogenic seizures was accompanied by an elevation of the Preyer (acoustic startle response) threshold, it was suggested that reduction of seizures was simply the result of noise-induced hearing loss: i.e., the magnitude of neural activity necessary 1 Supported K.R.H.
by NSF Grant No. GB 31921 and PHS
542 Copyright All rights
s
1975 by Academic Press, Inc. reproduction in any form reserved.
Grant No. NS 11565-01 to
AUDITORY
543
FUNCTION
to initiate audiogenic seizures, which is normally evoked by an electric bell, was not realized because of impaired auditory function, so that the animals were protected from seizures. The experiments reported below were designed to test this hypothesis with the use of auditory evoked potentials, the best method available to examine noise-induced hearing loss in laboratory mice. Hearing loss should be reflected in the auditory evoked potential by elevated absolute thresholds and depression of suprathreshold amplitudes (2, 3, 12, 13). Experiment 1 was a preliminary experiment in which our previous (8) study was replicated in every detail, except that instead of observing Preyer responses and audiogenic seizures, auditory evoked potentials were recorded. Experiments 2 and 3 used a different strain of mouse and were designed to provide a more controlled examination of the relationship between audiogenic seizures and noise-induced hearing loss as measured by Preyer thresholds, and thresholds and amplitudes of auditory evoked potentials. EXPERIMENT
1
Method Twenty C57BL/6 J mice were acoustically primed at age 16 days and tested at age 21 days, the 5 day lapse being optimum for the development of susceptibility to audiogenic seizures (4). On the day of testing, all subjects were anesthetized lightly with ether. Ten subjects (exposed group) were exposed to the sound of a 5 cm electric bell (mean intensity 114 db SPL) in a glass bell jar for 30 sec. Etherization was necessary to prevent seizures during this exposure to sound. The remaining ten subjects (control group) were also etherized but were not exposed to sound. Four hours later, all subjects were anesthetized with sodium pentobarbital (Nembutal 1.5 pg/g of a 50 mg/db solution) for recording of auditory evoked potentials. This was the first departure from our previous experiment in which subjects were tested for Preyer thresholds and audiogenic seizures instead of being anesthetized, In line with our previous data, auditory evoked potentials of the exposed group should be associated with a state of low susceptibility to seizures, whereas auditory evoked potentials from the control group would be those obtained from highly susceptible animals. The method for obtaining auditory evoked potentials has been described in detail (18). Briefly, anesthetized subjects were placed in a stereotaxic headholder which did not utilize earbars. The skull was exposed, and bipolar steel electrodes were lowered through the soft skull into the inferior colliculus. The contralateral ear was presented with a lOkHz-20kHz noise band (25 msec bursts, 1 msec rise and decay, 4 pulses/set) which
544
WILLOTT,
HENRY
AND GEORGE
was controlled without producing transients. Sound was delivered through a T-shaped metal tube, with one opening going to the subject’s ear and the other to the microphone of a Bruel and Kjoer SPL meter. This provided a close approximation of the sound pressure at the subject’s ear. Auditory evoked potentials were amplified and relayed to an oscilloscope. The subject’s absolute threshold for auditory evoked potentials was initially measured. This parameter, defined as the lowest intensity which evoked a visible (2 pv) response on the oscilloscope, provided an estimate of absolute auditory sensitivity. Then the intensity of the acoustic stimulus was increased in 10 db steps to 100 db, the maximum intensity our system could deliver without disortion. At each 10 db increment, the peak-topeak auditory evoked potentials of the amplitude was recorded. RESULTS Figure 1 depicts input-output functions for the exposed and control groups. The absolute auditory evoked potential threshold was elevated by 5.8 db in the exposed group [t( 18) = 2.47, P < 0.051, undoubtedly reflecting the threshold shift which typically accompanies exposure to intense sounds. At moderate intensities, there appears to be some depression of the auditory evoked potential amplitude in the exposed curve, but the greatest differences are not quite statistically significant with a one-tailed t-test. It is quite evident that at the highest intensities there was no difference in auditory evoked potential amplitude. The results of this preliminary experiment are clear-cut. While there was an elevation of the absolute auditory evoked potential threshold,
FIG. 1. Input-output
functions for C57BL/6J
mice of Expt. 1.
AUDITORY
545
FUNCTION
suprathreshold amplitudes showed little if any effects of exposure to the intense noise, despite the fact that such exposure reduces audiogenic seizures and elevates Preyer thresholds. In order to adequately interpret these results, however, it was necessary to provide greater stimulus control for two reasons. First, in Expt 1 an electric bell was used for noise exposure, while a 10-20 kHz noise band was used for auditory evoked potentials. This noise band includes the frequencies most effective in inducing audiogenie seizures (7) but the bell could have affected responses to very high or low frequencies which could conceivably influence seizures. Such effects could have gone undetected by auditory evoked potentials evoked by the noise band. Second, the maximum intensity we were able to provide for auditory evoked potentials was 100 db SPL, whereas the electric bell provided a mean intensity of 114 db. Furthermore, the aperiodicity and irregular energy distribution of the electric bell could be important factors. It was therefore necessary to perform the following experiments in which these variables were more tightly controlled. EXPERIMENT
2
Method This experiment was designed to minimize differences in the acoustic stimuli used for noise exposure, for inducing seizures, for measuring Preyer thresholds, and for obtaining auditory evoked potentials. Also, DBA/2J mice were used as subjects to eliminate the need for priming, as well as to extend our research to another strain of mouse. This strain is innately highly susceptible to audiogenic seizures, allowing a less intense sound to be used to induce seizures. New and improved equipment also aided the present and following experiments. Thirty-nine 23-24 day-old DBA/2 J mice served as subjects. On the day of testing, all subjects were lightly anesthetized with ether, again, to protect those which were exposed to noise from having seizures. The exposed group was placed in a 20 X 15 cm diameter plexiglass cylinder placed upright on the floor of a sound proofed box. At the top of the cylinder an Altec 802 tweeter housed in a 17 X 19 X 19 cm box provided the acoustic stimulus, which was 90 sec. duration of a 10-20 kHz noise band at a mean intensity of 120 db SPL (sound pressure level). This was ascertained by initially measuring at the position of the mouse in 1 kHz bands. The SPL was 130 db at 10 kHz and dropped linearly to 110 db at 20 kHz. This closely approximated the spectral and intensity characteristics measured at the ear of the mouse when auditory evoked potentials were obtained in Expt 3. The control group was treated identically, but not exposed to the noise. Fifteen to 20 min later, after recovery from the effects of ether,
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Preyer thresholds were obtained and subjects were tested for audiogenic seizures. For Preyer thresholds, either control (ten) or exposed (ten) subjects were replaced into the sound chamber. Preyer responses were elicited by brief bursts of the same 10-20 kHz noise band (25 msec rise and decay time). Preyer threshold was obtained by varying the intensity of the noise bursts until the minimum level capable of eliciting the startle response was found. For each of the above subjects, immediately after the Preyer threshold was obtained, the intensity of the noise band was raised to 110 db SPL for 1 min to test for audiogenic seizures. An additional 19 subjects had been previously tested for seizures in a pilot study and their data are included here. For ‘every subject, the occurrence of each scccessive stage of audiogenie seizures-wild running, clonic seizure, tonic seizure, and death-was recorded, and latency to wild running was measured with a stopwatch. Results The results of Expt. 2 are presented in Table 1. The data clearly show an elevation of the Preyer threshold for the exposed group. This group had a mean Preyer threshold of 102 db SPL compared to 87 db for the control group [ t( 18) = 7.49, P < O.OOl] . The elevation of the Preyer threshold was comparable to that obtained from C57BL/6J mice in the previously cited study (8). Although that study used an electric bell for noise exposure, it used the same lo-20 kH z noise band for Preyer responses. Table 1 also shows that the exposed subjects were clearly protected from audiogenic seizures. For the exposed group, the mean latency to seizures (wild running stage) was increased by 3.1 sec. [ t(38) = 4.331, TABLE PREYER
THRESHOLDS
AND SEVERITY
1 OF AUDIOGENIC
SEIZURES
(EXPT
2)a
Parameter
Control
Exposed
Preyer threshold Mean latency to wild running Mean severity of seizureb Incidence of seizures Incidence of deaths
87 db 2.9 set 3.6 19 of 19 subjects 15 of 19 subjects
101 db 5.0 set 1.2 11 of 20 subjects 4 of 20 subjects
a Subjects were DBA/2J mice. The exposed group was subjected, thesia, to 90 set of a lo-20 kHz noise band, 15-20 min before data co&o2 group was treated identically, but not exposed to sound. b Severity was scored as: 1 = wild running; 2 = clonic seizure; 4 = death (5).
under ether was obtained. 3 = tonic
anesThe seizure;
AUDITORY
547
FUNCTION
P < O.OOl], and the mean severity of seizures was decreased to one-third that of controls [t(38) = 6.082, P < O.OOl]. Also, the incidence of seizures for the control group was 100% compared to 55% for exposed (x” = 8.725, P < O.OOl), and the incidence of lethal seizures was 79% for controIs compared to 20% for exposed subjects (x2 = 11,295, P < 0.001). EXPERIMENT
3
Method The final experiment was a replication of Expt. 2, except that instead of observing Preyer responses and audiogenic seizures, auditory evoked potentials were recorded. This allowed a determination of noise-induced hearing loss, as measured by auditory evoked potential parameters, which accompanied the elevated Preyer thresholds and reduced audiogenic seizures observed in Expt. 2. The main improvement in this experiment is that the same noise band was used for auditory evoked potentials as was used for acoustic exposure, Preyer responses, and audiogenic seizures. This markedly reduced the possibility of extraneous effects of noise exposure on the auditory system which might not be detected by auditory evoked potentials-a deficiency of Expt. 1. Twenty-six DBA/2J mice served as subjects. Exposed and control groups were treated the same as their counterparts in Expt. 2, except that auditory evoked potentials were recorded rather than measurement of Preyer thresholds and audiogenic seizures. The apparatus used to obtain auditory evoked potentials was similar to that described in Experiment 1, with several modifications. Most importantly, an Altec 802 tweeter replaced the Mustang Spheracon tweeter to provide the acoustic stimulus, allowing an intensity of 110 db to be delivered to the mouse’s ear in the stereotaxic apparatus. Thus, auditory evoked potentials in response to the intensity used to induce seizures could be recorded. Also, on-line computer averaging was obtained from ten mice with an Ortec signal averaging system, permitting more detailed analysis of auditory evoked potential waveform. The procedure for obtaining AEPs was also modified. Rather than obtaining full input-output functions, only absolute AEP thresholds and 110 db auditory evoked potentials were recorded. This reduced the possibility of auditory fatigue during auditory evoked potential measurement and limited suprathreshold measurement to the intensity used to induce seizures. The auditory evoked potential at this latter intensity should be most relevant to audiogenic seizures. An additional procedure was also added to examine possible differences in “fatigability” of the ears of exposed and control groups. Since seizures
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begin after a latency of several seconds and their time course may run from 30 to 60 set, it is important to know if auditory fatigue during seizures could lead to reduced magnitude of neural activity. That is, did the exposed animals have increased vulnerability to auditory fatigue which reduced the severity of seizures by decreasing neural input during the initial stages of seizures? To test this possibility, subjects were exposed to two additional 15 set periods of noise while in the stereotaxic apparatus. After the initial auditory evoked potential threshold and 110 db auditory evoked potential had been recorded, 15 set of the noise band at 110 db were given. The auditory evoked potential was recorded immediately after. Next, a second additional 15 set noise exposure was given, and another auditory evoked potential was then recorded. Thus, for both exposed and control groups, the following auditory evoked potential parameters were obtained : (a) an initial absolute auditory evoked potential threshold ; (b) an initial auditory evoked potential in response to 110 db ; (c) a second auditory evoked potential in responseto 110 db, but following an additional 15 set noise exposure ; and (d) a third 110 db following still another 15 set noise exposure. RESULTS For the exposed group the absolute auditory evoked potential threshold was 42.1 db compared to 23.2 db for the control group [t (24) = 3.447, P < 0.011, as seen in Tbble 2. This is consistent with the results of Expt. 1 and is indicative of the expected noise-induced threshold shift. However, the peak-to-peak amplitude of the initial auditory evoked potential at 110 db was not affected, as can be seen in Fig. 2. Furthermore, latency and waveform did not differ between the two groups. These results are clearly in accord with those of Expt. 1: whereas noise exposure was followed by TABLE AUDITORY
AEP AEP AEP AEP
threshold amplitude amp. after amp. after
EVOKED
POTENTIAL
2 PARAMETERS
FOR EXPT
Parameter
Control
(110 db) first additional exposure second additional exposure
23.2 db 74 flv 62 pv 58 /N
a Subjects were DBA/ZJ mice which had either been exposed of 110 db of a lo-20 kHz noise band 15-20 min before auditory ing. “Additional exposures” were 1.5 set of the same noise band were in the stereotaxis apparatus. ) P(C = E) < 0.01.
3a Exposed 42.1 db” 83 /N 65 /N 51 )bv
or not exposed evoked potential at 110 db while
to 90 set recordsubjects
AUDITORY
FIG. 2. Peak-to-peak
549
FUNCTION
AEP amplitude for DBA/ZJ
mice of Expt. 3.
an increase in absolute auditory evoked potential threshold, high intensity auditory evoked potential amplitudes were not affected. The results of the second and third 15 set noise exposure are also seen in Fig. 2. For both control and exposed groups, the two additional noise exposures were effective in reducing auditory evoked potential amplitude immediately after exposure. Analysis of variance for repeated measures showed both groups had significant reductions in auditory evoked POtential amplitudes [exposed: F (2.24 = 22.598, P < 0.001; control: F(2.24) = 6.225, P < 0.011 as a result of the additional exposures. Further analysis indicates that the greatest reduction of auditory evoked potential amplitude occurred following the first additional exposure for both control [t(l2) = 2.355, P < 0.05] and en-fiosed [t(12) = 4.534, P < 0.0005]. Although it appears from Fig. 2 that the curve for the exposed group has a steeper decline than that for the control group, the curves did not differ significantly as indicated by the lack of interaction between conditions (control, exposed) and additional exposures [F (2.48) = 2.279, P > 0.11. It appears from these data that the additional noise exposures were effective in reducing the amplitude of the 110 db auditory evoked potential, but there was little if any difference in the magnitude of this effect between control and exposed groups. Using this as our criterion, it appears that the exposed animals were not significantly more susceptible to auditory fatigue than the controls. DISCUSSION The present study set out to determine whether or not protection from audiogenic seizures could be accounted for by noise-induced hearing loss. The data indicate that the answer is not a simple one. The elevated
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Preyer thresholds and auditory evoked potential thresholds indicate that hearing loss did occur, whereas the auditory evoked potential amplitudes at seizure-inducing intensity provides no evidence of hearing loss. It is pertinent to first examine the nature of noise-induced hearing loss in terms of the auditory evoked potential threshold, Preyer threshold, and auditory evoked potential amplitude and then discuss their relevance to audiogenic seizures. The elevation of absolute auditory evoked potential threshold indicates that hearing loss was evident at low intensities. Such threshold shifts have been demonstrated at various levels of the auditory system following exposure to noise (2, 3, 12, 13, 17, 18). Furthermore, elevation of the thresholds for auditory evoked potentials has been directly associated with behaviorally measured hearing loss (12). Elevation of the Preyer threshold has also been reliably associated with exposure to intense noise and with conductive hearing loss (8, and an unpublished study in our laboratory). It seems quite certain that the consistent elevation of the Preyer threshold following noise exposure in the present study is a function of hearing loss at moderate intensity. The amplitude of the auditory evoked potential at high intensities of stimulation has also been shown to be depressed by exposure to intense sound. This has been demonstrated in both the peripheral and central auditory pathway (2, 12, 13, 17, 18). In fact, Expt. 3 showed that exposure to additional periods of noise resulted in depression of the 110 db auditory evoked potentials immediately after exposure (Fig. 2). Depression of the auditory evoked potential amplitude, therefore, seems to be a reliable indicator of hearing loss at higher intensities. The lack of effect on the auditory evoked potentials of the exposed groups of Expt. 1 and 3 therefore indicates that the subjects rapidly recovered from whatever high intensity hearing loss they may have suffered. If then, the auditory evoked potential threshold, Preyer threshold, and auditory evoked potential amplitude are all reliable indices of hearing loss, one must conclude that hearing loss was nonlinear in the present study. That is, there was a loss at low and moderate intensities auditory evoked potential and Preyer threshold elevations) but no diminution of high intensity responses (auditory evoked potential amplitudes). This type of relationship has in fact been reported for both animal auditory evoked potential and human loudness experiments. In animals, Rosenblith et al. (13) found that the effects of high intensity sound exposure were greater for low intensity than for high intensity responses, as indicated by the cortical auditory evoked potential of cats. Recently, Pugh et al. (12) showed that, while the suprathreshold eighth nerve potential was initially depressed by sound exposure, its rate of growth increased rapidly despite
AUDITORY
FUNCTION
551
threshold elevation. Similar effects have been demonstrated with auditory evoked potential in mice (6, 14, 17, 18). With human subjects, Davis et al. (1) showed that noise exposure elevated absolute thresholds of hearing but had little effect on the loudness of higher intensity sound. A similar effect was reported by Small and Canakl (15), who found that temporary threshold shifts were much greater than loudness shifts following sound exposure. McPherson et al. (11) observed that loudness and threshold shifts may recover at different rates. In light of such evidence, it seems quite reasonable that the high intensity responsiveness of the exposed groups recovered by the time of testing for audiogenic seizures. The slower recovering threshold and Preyer responses,however, still indicated hearing loss. If auditory evoked potential amplitudes at seizure-inducing intensities are not depressed by the noise exposures used in the present study, what is the mechanism of protection from seizures? Clearly, a general reduction of the magnitude of neural activity evoked by sound, as measured by the auditory evoked potential, cannot account for the inhibition of seizures. Such a reduction of seizures should be associated with decreased auditory evoked potential amplitude. It is more likely that the mechanism of protection from audiogenic seizures may instead involve disruption of patterns of neural activity normally associated with seizures. It is most certain that different populations of receptors and neurons are functional before and after exposure to noise. Some components, those most vulnerable to trauma, are eliminated by auditory fatigue or damage. The loss of such responsesare apparently most evident in such measuresas absolute threshold and Preyer threshold. In light of the present findings, it seems that those elements of the auditory system which have been so affected are necessary for the initiation of audiogenic seizures. What processes are involved are at present unknown, but it does seemthat the specific pattern rather than magnitude of neural activity is most important in protection from seizures. This interpretation is consistent with a previous study from our laboratory which demonstrated protection from seizures by physical restraint of movement ( 15), where it seemedmost likely that disruption of normal patterns of neural activity was involved. With regard to the relationship between auditory evoked potential amplitude of the susceptibility to audiogenic seizures, the results of the present investigation provide evidence that the two are not closely related. Highly susceptible (control) mice and low susceptible (exposed) mice had auditory evoked potential amplitudes which did not differ. These results are in agreement with several recent reports, which also failed to find this relationship (9, 10, 18). Such findings do not, however, support earlier hypotheses which associatedacoustic priming for audiogenic seizures
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AND
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with “disuse supersensitivity” (6, 14). These studies demonstrated that, following priming, auditory evoked potential amplitudes became elevated as susceptibility to audiogenic seizures developed. It was hypothesized that the high auditory evoked potential amplitude indicated supersensitivity of the nervous system which resulted in susceptibility to audiogenic seizures. The present study and the other studies cited indicate that, whatever might be the cause of auditory evoked potential amplitude increased following priming, it is not responsible for audiogenic seizures. It is evident that an explanation of audiogenic seizures must go further than simply considering the amplitude of neural activity. In summary, the data reported here demonstrate that the effects of exposing the auditory system to noise are more complex than one might suppose, at least in regard to the inhibition of audiogenic seizures. The results of two different experimental designs using two strains of mouse suggest that specific patterns of neural activity in the auditory system are crucial in the initiation of audiogenic seizures. Exposure to noise may interfere with the functioning of elements in the auditory system which are most vulnerable to damage and thereby disrupt patterns of neural activity which normally are necessary for seizures. The fact that disruption of these patterns results in protection from audiogenic seizures may carry some implication for protection from other types of seizures. REFERENCES H., C. T. MORGAN, J. E. HAWKINS, R. GALAMBOS, and F. W. SMITH. 1950. Temporary deafness following exposure to loud tones and noise. Acta
1. DAVIS,
oto-Luryngol. 2. DAVIS, H., 3. 4. 5.
6.
suppz.
88.
et al. 1953. Acoustic trauma in the guinea pig. J. Acoust. Sot. Amer. 25 : 1180-1189. GISSELSSON, L., and H. SORENSEN. 1960. Auditory adaptation and fatigue in cochlear potentials. Actu Oto-Laryngol. 50: 391-405. HENRY, K. R. 1967. Audiogenic seizure susceptibility induced in C57B1/6J mice by prior auditory exposure. Science 158: 938-940. HENRY, K. R., and R. E. BOWMAN. 1969. Effects of acoustic priming on audioand chemoconvulsive seizures. J. Camp. Physiol. genie, electroconvulsive, Psychol. 67 : 401-406. HENRY, K. R., and M. SALEH. 1973. Recruitment deafness: functional effect of priming induced audiogenic seizures in mice. J. Comb. Physiol. Psychol. 84:
430-43s. 7. HENRY, K.
teristics
R., K. A. THOMPSON, and R. F. BOWMAN. 1971. Frequency characof acoustic priming by audiogenic seizures in mice. Exp. Neural.
31: 402-407. 8. HENRY, K. R.,
and J. F. WILLOTT. 1972. Unilateral inhabition of audiogenic seizures and Preyer reflexes. Nature (London) 240 : 481-482. 9. MCGINN, M. D., and K. R. HENRY. Acute versus chronic acoustic deprivation: Effects of auditory evoked potentials and seizures. Dev. Psychobiol. (in press).
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10. MCGINN, M. D., J. F. WILLOTT, and K. R. HENRY. 1973. Effects of conductive hearing loss on auditory evoked potentials and audiogenic seizures in mice. Nature [New Biol.] 244: 255-256. 11. MCPHERSON, D., D. FRANK, and C. V. ANDERSON. 1970. Relation of temporary loudness shift to temporary threshold shift. J. Acoust. Sot. Amer. 49: 11951202. 12. PUGH, J. E., M. R. MILTON, and D. J. ANDERSON. 1974. Cochlear electrical activity in noise-induced hearing loss. Arch. Otolar. 100 : 36-40. 13. ROSENBLITH, W. A., R. GALAMBOS, and I. HIRSH. 1950. The effect of exposure to loud tones upon animal and human responses to acoustic clicks. Scieme 111: 569-571. 14. SAUNDERS, J. C., G. R. BOCK, R. JAMES, and C. S. CHEN. 197.2. Effects of priming for audiogenic seizures on auditory evoked responses in the cochlear nucleus and inferior colliculus of BALB/c mice. ExD. Neural. 37: 38-394. 15. SMALL, A., and J. A. CANAHL. 1965. Loudness changes in a forward-masking stimulus paradigm. J. Acoust. Sot. Amer. 38: 928. 16. WILLOTT, J. F. 1974. Protection from lethal audiogenic seizures in mice by physical restraint of movement. Exp. Neural. 43: 359-368. 17. WILLOTT, J. F. Some effects of exposure to intense acoustic stimulation on input-output functions in mice. J. Aud. Rex. (in press). 18. WILLOTT, J. F., and K. R. HENRY. 1974. Auditory evoked potentials: developmental changes of threshold and amplitude following early acoustic trauma. J. Camp. Physiol. Psychol. 86: 1-7.
