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Ultrasonic electrocochleography in guinea pig
Hemng
Research,
17 (1985)
143-151
143
Elsevier
HRR 00580
Ultrasonic
electrocochleography
Kenji Ohyama, Depcrrtmeni
of Otola~ngolog~,
(Received
12 January
Jun Kusakari School of Medtcine,
1984; revised manuscript
in guinea pig
and Kazutomo
Tohoku
Unrr~ersrty. Serryo
received 2 January
Kawamoto -rho
I
1985; accepted
I,
Sendai,
8 January
YXO,Jupm
1985)
In order to obtain information about how ultrasonic stimuli are perceived (USP) in man, guinea pig cochleae were stimulated by bone conduction with frequencies (98.8 and 143.5 kHz) above the normal auditory field of this animal. The cochlear potentials recorded consisted of CM, SP and AP originating from the basal turn of the cochlea. and were found to be influenced by asphyxia, administration of ethacrynic acid, hypothermia and change of interstimulus interval. In addition. in kanamycin-treated animals the mean AP amplitude decreased to about one fourth of the normal value. and the mean AP latency increased significantly. These findings suggest that there is no special sense organ for the detection of LISP but that such sounds activate hair cells in the basal turn of the cochlea. guinea
pig. electrocochleography,
ultrasonic
perception,
hypothermia.
Introduction While the upper frequency limit of human hearing is generally considered to be around 20 kHz, the presence of a curious phenomenon called ultrasonic perception (USP) [l] has been repeatedly reported upon in the last few decades. Although various studies have been conducted to examine this phenomenon, its mechanism has not yet been fully elucidated [1,3,5,7,8,10,11,16]. A recent report showed that the human auditory system responds to ultrasonic stimuli presented by bone conduction [13]. In order to identify the site within the cochlea which is stimulated by ultrasound, ultrasonic vibration stimuli were presented to guinea pig cochleae, and electrocochleograms were recorded under several conditions. This paper reports this new approach to the elucidation of USP and discusses the mechanism of this phenomenon. Materials and Method 21 albino guinea pigs weighing 230-500 g each and with active Preyer pinna reflexes were used. 037X-5YS5/85/$03.30
” 1985 Elsevier Science Publishers
kanamycin
ototoxicity
Each animal underwent tracheostomy under general anesthesia using intraperitoneally administered pentobarbital sodium (Nembutal, Abbott Laboratories, 28 mg/kg). The animals were then given suxamethonium chloride (Relaxin, Kyorin. 50 mg/kg) intramuscularly and artificially ventilated. The left tympanic bulla was exposed via a ventrolateral approach. Acoustic stimuli were generated by a closed, dynamic earphone system (Dana Japan DA-SD) and delivered through a hollow ear bar. The driving signal was a 90 ps rectangular pulse (click) of alternating polarity, with a maximum level of 106 dB SPL, and the signal delay (the interval from the trigger to the onset of the response) was 0.61 ms. Ultrasonic vibration stimuli were generated by the triggered function generator system (NF FG121B, Leader LFG-1300) and delivered to the animals through an ultrasonic transducer which was pressed against the neck muscles ventral to the cochlea. The waveform of the signal was a sinusoidally enveloped random phase pip of 98.8 kHz and 0.5 ms duration (Fig. la). This waveform was selected to minimize the undesirable influence of transient distortion products in the response.
B.V. (Biomedical
DiGsion)
a
-
/
Fig. 2. Diagram of the experimental system. Differential cordings (SV-ST) were also made in some cases.
Fig. 1. (a) Waveform of the stimulus. Sinusoidally enveloped pip of 98.8 kHz or 143.5 kHz was selected to minimize undesirable transients. (b) Electrodynamic ultrasonic transducer used in this study had a resonant frequency of 98.8 kHz and resonant impedance of 590 52. (Scale in centimeters.)
The intensity of the ultrasonic stimulus was taken to be the peak-to-peak voltage of the signal at the amplifier output to the transducer, using a maximum peak-to-peak voltage of 20 V as the reference (0 dB). In this case, the delay time was negligible. The ultrasonic transducer, shown in Fig. lb, had a lowest resonant frequency of 98.8 kHz and a resonant impedance of 590 0. Another transducer resonating at 143.5 kHz was also used in several cases. Cochlear potentials were recorded through a silver-wire electrode placed on the ventral edge of the round window, with reference and ground electrodes being located on the denuded neck muscles. In some animals, differential recordings were obtained by inserting needle electrodes through small burr holes into the Scala vestibuli (SV) and the Scala tympani (ST) of the basal and second turns of the cochlea [6]. The responses were amplified with a Grass P-18 amplifier and then averaged 32 times with a signal processor (San-Ei, 7T07A). A diagram of the experimental system is shown in Fig. 2. The responses evoked by clicks and ultrasound
re-
were recorded in 12 control animals, and also in animals treated with ethacrynic acid, kanamycin, or hypothermia. A single dose of 40-50 mg/kg of ethacrynic acid (Edecrin; Merck, Sharp & Dohme) was given through an indwelling catheter in a neck vein to each of four animals. Nine animals were made kanamycin toxic by subcutaneous injection of kanamycin sulfate (Meiji, 450 mg/kg) daily for a week, 14-46 days before testing. The ECG was monitored throughout the experiment, and body temperature was monitored by a rectal probe and maintained between 36.5 and 38.5”C except for the, hypothermia portion of the study. Results Normal response
In 12 animals clear, reproducible responses were obtained to ultrasonic stimulation. The ultrasonic-evoked response (UER) waveform was composed of an initial positive deflection, followed by two or three sharp negative peaks (Fig. 3a), and closely resembled the click-evoked responses (CER) shown in Fig. 3b. When the stimulus intensity was lowered the amplitude of the UER decreased and finally disappeared at about - 20 to - 30 dB (2.0-0.6 V peakto-peak) (Fig. 4). The mean value of the stimulus intensities for the minimal detectable level was 1.71 V peak-to-peak (- 21.4 dB). When the stimulus intensity was reduced the latency of the first negative peak increased slightly perhaps as the result of a delay in the time to reach effective stimulus level [4].
145
trigger .
> 0’ 0 m
Frg. 3. (a) The ultrasonic-evoked response (UER) is composed of initial positive and succeeding negative deflections. They are identified to be SP and AP, respectively. SP amplitude is measured from the baseline to the initial peak, and AP amplitude is measured from the initial peak to the first negative dip. (b) The click-evoked response (CER) from the same ear. UER and CER resemble each other except in the prominent SP of the former.
The interstimulus interval (ISI) was found to influence the UER waveform. By decreasing the ISI from 200 to 2.5 ms, the amplitude of the first negative peak was reduced by 50 or 60% but no change was noted in the initial positive deflection (Fig. 5). Asphyxic anoxia, induced by stopping the ventilator, produced substantial changes in the UER waveform. However, the recovery process began immediately after the restoration of artificial ventilation (Fig. 6). The time courses of the amplitudes of the two components of the response seen after 2 min of asphyxia are shown in Fig. 7a and b. Considering the physiology of the cochlea, these negative peaks with adaptation phenomena are probably cochlear action potentials (AP). In addition, differential electrodes in the basal turn of the cochlea detected another form of response. With the onset of asphyxia this potential changed in much the same way as did the initial positive deflection of the
a 0
-- 6.0
-16.5
+* 1 -22.5
v
(dB r~
20V~)
d’
l0i
_ [ees
mt
HII]
I
50
-40
30
-20
IQter7,sl
Fig. 4. (a) Strmulus attenuation causes a decrease in UER amplitude. and UER disappears at about input&output functions of the AP in the UER (- - - - -) and CER ( -). The curves of the UER are the CER. (Responses from the same ear are plotted with the same symbols.)
10
ti,
!c1B!
~20
to -30
far
steeper
than
0
dB. (b) The are
those
of
100
50
25
goxl----20
10 t-4
2 ms
5
>s
H
(m set)
Fig. 6. UER changes induced by 2 min asphyxia and afterward. Complete recovery takes place about 7 mm after resumption of respiration (120 t).
2 mssc Fig. 5. Effect of reducing the interstimulus interval (ISI) upon the UER. The initial positive component shows no particular change, but the following negative components show remarkable adaptation which is considered to be neural in origin.
UER recorded at the round window, although those two responses were opposite in polarity (Fig. 7~). No response component comparable to that from the basal turn was detected from the second turn of the cochlea (Fig. 8). It may be concluded that the component of the response from the basal turn is the summating potential (SP). Direct observation of the differentially recorded responses from the basal turn revealed a microphonic-like component with an amplitude of about 200 PV (peak-to-peak) to be overlapping the SP. This component had the same frequency as the stimulus signal and exhibited remarkable lability in response to anoxia (Fig. 9). Thus it was identified as a cochlear microphonic potential (CM). No
CM was recorded cochlea.
from
the second
turn
of the
Effect of ethacrynic acid The UER were recorded from four guinea pigs before and after intravenous injection of ethacrynic acid. In the first animal, 50 mg/kg of ethacrynate reduced the SP component of the UER to zero within 9 min, inverted the polarity of the response between 9 and 11 min, and caused it to reach a low point at about 11 rnin after administration. Then a slow recovery process occurred and the SP, after a transient overshoot, returned to the original level. The second animal was given 40 mg/kg of ethacrynate, and SP and AP amplitudes were reduced to 40% and 608, respectively, of the preadministration values within 25 min of administration. In the third animal, administration of 50 mg/kg ethacrynic acid led to rapid disappearance of potentials; no recovery of AP was noted within
147
AP
is-
---
-ie
Fig. 7. Effects of asphyxia on amplitudes of AP (a). SP (b), and DIF-SP (c) from the basal turn of the guinea pig cochlea. Closed triangle (A) marks resumption of respiration; the transient overshoot about 446 min after the onset of asphyxia is characteristic.
a
b
Fig. 8. Representative traces from the basal turn (a) and from the second turn (b) of the guinea pig cochlea showing differen-
Fig. 9. Direct observation of differentially recorded potentials. An ultrasonic signal of 98.8 kHz was used as the stimulus (upper trace). The CM response contained overlapping AP and SP (middle trace). There was an obvious reduction of CM amplitude after 2 min of asphyxia (bottom trace).
4 h. A similar effect was observed in the last animal, in which the differentially recorded SP (SV-ST) disappeared rapidly. The effects of ethacrynic acid on SP and AP amplitudes are shown in Fig. 10. In general, administration of high doses of ethacrynate produced a rapid depression of AP and SP within a few minutes, followed by a recovery process which took several hours. This phenomenon is quite similar to the effect of ethacrynate upon the acoustically evoked AP and SP [2]. Effect of hypothermia In three animals the effect of body temperature upon the CER and the UER was examined, using maximal stimulus levels. For both responses, hypothermia led to decreased amplitude, increased latency, and broadening of the waveform, as shown in Fig. Il. Both responses were affected by hypothermia in much the same way. Other authors have investigated the effect of body temperature on AP latency, and determined that the main site of this phenomenon is the afferent hair cell-neuron synapse [15]. The results tially recorded SPs and how they altered during and after 2 min of asphyxia. No significant SP was detectable in the response from the second turn.
-iBo--*
240
2
SP __ __.___-.--------A---v__
120
160
240
4
Fig. 10. Ethacrynate caused SP and AP to decrease rapidly in four guinea pigs (1,2,3,4) within a few min after injection. SP recovered more rapidly than did AP.
of the present tion does not any other site cells just like sounds.
study suggest that ultrasonic perceptake place at the cochlear nerve, nor central to it, but in the cochlear hair the perception of normal, audible
Responses in the kanamycin-treated animals Kanamycin
(KM) treatment
reduced
the ampli-
tude of the UER and increased AP latency IO varying degrees. The mean value of the amplitude of the response to maximum ultrasonic stimulation was 127.1 pV, which was about a quarter of the normal value (560.4 pV). while AP latency at maximum stimulation was 1.21 f 0.14 ms, significantly greater than normal (0.95 + 0.06 ms). This difference in latency remained almost constant at all stimulus levels (Fig. 12). Discussion Although ultrasonic perception (USP) in man is not always well known even to audiologists or physiologists, its existence is now beyond doubt. However, lack of appropriate methodology to investigate this phenomenon objectively has precluded the conducting of a detailed physiological study of the mechanism by which this occurs. Thus, it seems extremely important to develop an animal model so that this subject can be investigated more fully. Behavioral studies have shown that the uppermost frequency of the guinea pig auditory field lies around 50 kHz [9]. The signal frequencies used in the present experiment (98.8 and 143.5 kHz) are, therefore, obviously ‘ultrasonic’ to the guinea pig. Nevertheless, the results of the present study showed that such signals evoke cochlear potentials just as do sounds within the auditory field. This is considered to be objective evidence that guinea pigs have ultrasonic perception. Although several hypotheses have been proposed to explain the perception of ultrasonic stimuli (Table I, [S]), the mechanism by which such perception occurs still has not been determined. In the present study, ultrasonic stimuli resulted in clear SP and CM, in addition to AP. Furthermore, these potentials were affected by anoxia, hypothermia, and the administration of kanamycin or ethacrynic acid. These results strongly suggest that the SP and CM recorded in the present experiment were not artifacts but rather evoked by ultrasound. Therefore, it is reasonable to presume that the primary site of ultrasonic action may be in sensory hair cells of the basal turn of the cochlea rather than in the cochlear nerve. In a recent report [13], AP evoked by ultra-
149
C
b UER
CER 2
Fig. Il. (a) Representative traces of UER and CER during body cooling (hypothermia). Amplitude decrease, latency increase, and decreased sharpness of AP are evident in both the UER and CER. The effect of body temperature on AP latency of UER is shown in b. and of body temperature on AP latency of CER in c; there was no significant difference between these responses.
sound were compared with those evoked by clicks. The former were found to have a steeper input-output function and poorer adaptation to a
reduction in ISI than the latter. These results indicated that auditory potentials evoked by ultrasound are similar to those evoked electrically by
b
a
. -28
I
-Ifi
Intensity
[dBl*
direct stimulation of the cochlear nerve. However, the results obtained in the present study, especially the presence of CM and SP evoked by ultrasonic stimuli and the alterations in AP
I
HYPOTHESES
ON ULTRASONIC
PERCEPTION
I. Utrasonic perception as a result of inadequate stimulation particular sections of the hearing system: (1) Direct stimulation of co&ear nerve fibers. (2) Stimulation of organ of Corti in some different manner.
of
2. Ultrasonic perception as a result of adequate stimulation of the inner ear: (1) The existence of rudimentary ultrasonic receptors. (2) The generation of subharmonic vibrations in the range of audible frequencies through non-linearities in the transmission path of ultrasonics. (After
Dieroff
and Ertel [8], partly
modified.)
-ia
Intensity
Fig. 12. (a) AP input-output UER functions of normal (-; Kanamycin treatment reduces the AP amplitude to about a fourth kanamycin-treated animals. AP latency is considerably elongated
TABLE
--i&3
idR1°
n = 12) and kanamycin-treated (- - - - - -; n = 9) animals. of normal. (b) AP intensity-latency UER functions of normal and in the latter. (Vertical bars indicate standard error of the mean.)
latency noted with hypothermia, permit us to discard the cochlear nerve stimulation theory. This theory may be called the ‘inadequate stimulation’ hypothesis. It is also evident that the subharmonits theory, according to which the sound heard during exposure to ultrasonic stimuli is merely a distortion product within the auditory field, is not adequate to explain this phenomenon. Deatherage et al. observed that the pitch of the sound heard when ultrasonic stimulation is presented by bone conduction is the highest pitch which a subject can hear [7]. Then Dieroff and Ertel showed that ultrasonic stimuli are always determined to be in the frequency range 13-16 kHz, independent of the stimulus frequency (20-100 kHz) [8]. This shows the poor frequency resolution of this sensation. In addition, we previously reported upon the extremely prominent maskability of a bone-conducted ultrasonic signal
151
upon an audible sound above 5 kHz [12]. Such masking is clearly an exception to the general rule, are not effective at masking that is. “maskers lower frequency probe tones”. The most appropriate explanation for this latter phenomenon seems to be that the ultrasonic vibration stimulates the basal portion of the organ of Corti just enough to permit perception of a higher-frequency audible sound. This concept is not particularly new. In 1954 Deatherage et al. [7] described a similar mechanism which they called ‘diplacusis’, by which direct stimulation of the basal end of the cochlea led to perception of a sound. Data on the absolute thresholds for sonic and ultrasonic bone-conducted frequencies measured in human subjects by Corso [5] also support such a mechanism of inadequate stimulation. In the latter study an absolute threshold curve was obtained which showed a very marked rise (about 50 dB/octave) in the 14-20 kHz region, and a far more moderate slope in the ultrasonic region. This led to speculation that there may be a difference in the way sonic and the ultrasonic stimuli are perceived in the cochlea. It is impossible to conclude from the results of the present study whether ultrasound directly stimulates the hair cells or whether the basilar membrane is caused to vibrate first. As shown in Fig. 4b, the input-output curve of the AP evoked by ultrasound is steeper than that evoked by an audible sound. One possible explanation for this difference may be that the guinea pig ear normally recruits in these high-frequency regions. Recruitment of the whole-nerve AP input-output function, as may be found in some pathological ears, is attributed to loss of the tip of the single-unit tuning curve at the characteristic frequency (CF) [14]. If it is assumed that the sharp tuning mechanism does not function in ultrasonic perception, this difference can be explained easily. The results of the present study suggest that the mechanism by which hair cells are stimulated by ultrasound is different from that by which they are stimulated by audible sound. Thus, ultrasonic perception, at least in guinea pigs, is due to the activation of hair cells in the basal turn of the cochlea in quite a different manner from the way
in which they are activated the auditory field. Further clarify this phenomenon.
by normal sound within studies are necessary to
References 1 Abramovich, S.J. (1978): Auditory perception of ultrasound in patients with sensorineural and conductive hearing loss. J. Laryngol. Otol. 92, 861-867. 2 Aran, J.-M. and Charlet de Sauvage. R. (1977): Evolution of CM. SP and AP during ethacrynic acid intoxication in the guinea pig. Acta Otolaryngol. 83. 1533159. 3 Bellucci, R.J. and Schneider, D.E. (1962): Some observations on ultrasonic perception in man. Ann. Otol. Rhinol. Laryngol. 71, 719-726. 4 Charlet de Sauvage. R., Cazals, Y.. Erre. J.-P. and Aran. J.-M. (1983): Acoustically derived auditory nerve action potential evoked by electrical stimulation: An estimation of the waveform of single unit contribution. J. Acoust. Sot. Am. 73, 616-627. 5 Corso. J.F. (1963): Bone-conduction thresholds for sonic and ultrasonic frequencies. J. Acoust. Sot. Am. 35. 173881743. 6 Dallas. P.. Schoeny, Z.G. and Cheatham. M.A. (1972): Cochlear summating potentials: descriptive aspects. Acta Otolaryngol. Suppl. 302, 1-46. 7 Deatherage. B.H., Jeffress, L.A. and Blodgett. H.C. (1954): A note on the audibility of intense ultrasonic sound. J. Acoust. Sot. Am. 26, 582. 8 Dieroff. H.G. and Ertel, H. (1975): Some thoughts on the perception of ultrasonics by man. Arch. Otorhinolaryngol. 209, 277-290. 9 Heffner, R., Heffner, H. and Masterton. B. (1971): Behavioral measurements of absolute and frequency-difference thresholds in guinea pig. J. Acoust. Sot. Am. 49. 1888-1895. im Ultraschall10 Kietz. H. (1951): Hiirschwellenmessung gebiet. Acta Otolaryngol. 39. 183-187. 11 Kunze, W. and Kietz. H. (1949): Uber Horempfindungen im Ultraschallgebiet bei Knochenleitung. Arch. Ohr. Nas. Kehlkopfheilkd. 155, 6833692. K. and 12 Ohyama, K., Kusakari. J., Takasaka, T., Kawamoto. Yuasa. R. (1980): Investigation of human ultrasonic percepnon. Audiology (Japan) 23, 297. 13 Ohyama, K.. Kusakari, J., Takasaka, T., Kawamoto, K. and Yuasa. R. (1982): Cochlear and brainstem response evoked by ultrasonic vibration. Audiology (Japan) 25, 79-83. 14 Gzdamar, 0. and Dallas, P. (1976): Input-output functions of cochlear whole-nerve action potentials: Interpretation in terms of one population of neurons. J. Acoust. Sot. Am. 59, 143-146. auditory adaptation. II. 15 Prijs, V.F. (1980): On peripheral Companson of electrically and acoustically evoked action potentials in the guinea pig. Acustica 45. l-13. 16 Pumphrey, R.J. (1950): Upper limit of frequency for human hearmg. Nature (London) 166, 571.
Research,
17 (1985)
143-151
143
Elsevier
HRR 00580
Ultrasonic
electrocochleography
Kenji Ohyama, Depcrrtmeni
of Otola~ngolog~,
(Received
12 January
Jun Kusakari School of Medtcine,
1984; revised manuscript
in guinea pig
and Kazutomo
Tohoku
Unrr~ersrty. Serryo
received 2 January
Kawamoto -rho
I
1985; accepted
I,
Sendai,
8 January
YXO,Jupm
1985)
In order to obtain information about how ultrasonic stimuli are perceived (USP) in man, guinea pig cochleae were stimulated by bone conduction with frequencies (98.8 and 143.5 kHz) above the normal auditory field of this animal. The cochlear potentials recorded consisted of CM, SP and AP originating from the basal turn of the cochlea. and were found to be influenced by asphyxia, administration of ethacrynic acid, hypothermia and change of interstimulus interval. In addition. in kanamycin-treated animals the mean AP amplitude decreased to about one fourth of the normal value. and the mean AP latency increased significantly. These findings suggest that there is no special sense organ for the detection of LISP but that such sounds activate hair cells in the basal turn of the cochlea. guinea
pig. electrocochleography,
ultrasonic
perception,
hypothermia.
Introduction While the upper frequency limit of human hearing is generally considered to be around 20 kHz, the presence of a curious phenomenon called ultrasonic perception (USP) [l] has been repeatedly reported upon in the last few decades. Although various studies have been conducted to examine this phenomenon, its mechanism has not yet been fully elucidated [1,3,5,7,8,10,11,16]. A recent report showed that the human auditory system responds to ultrasonic stimuli presented by bone conduction [13]. In order to identify the site within the cochlea which is stimulated by ultrasound, ultrasonic vibration stimuli were presented to guinea pig cochleae, and electrocochleograms were recorded under several conditions. This paper reports this new approach to the elucidation of USP and discusses the mechanism of this phenomenon. Materials and Method 21 albino guinea pigs weighing 230-500 g each and with active Preyer pinna reflexes were used. 037X-5YS5/85/$03.30
” 1985 Elsevier Science Publishers
kanamycin
ototoxicity
Each animal underwent tracheostomy under general anesthesia using intraperitoneally administered pentobarbital sodium (Nembutal, Abbott Laboratories, 28 mg/kg). The animals were then given suxamethonium chloride (Relaxin, Kyorin. 50 mg/kg) intramuscularly and artificially ventilated. The left tympanic bulla was exposed via a ventrolateral approach. Acoustic stimuli were generated by a closed, dynamic earphone system (Dana Japan DA-SD) and delivered through a hollow ear bar. The driving signal was a 90 ps rectangular pulse (click) of alternating polarity, with a maximum level of 106 dB SPL, and the signal delay (the interval from the trigger to the onset of the response) was 0.61 ms. Ultrasonic vibration stimuli were generated by the triggered function generator system (NF FG121B, Leader LFG-1300) and delivered to the animals through an ultrasonic transducer which was pressed against the neck muscles ventral to the cochlea. The waveform of the signal was a sinusoidally enveloped random phase pip of 98.8 kHz and 0.5 ms duration (Fig. la). This waveform was selected to minimize the undesirable influence of transient distortion products in the response.
B.V. (Biomedical
DiGsion)
a
-
/
Fig. 2. Diagram of the experimental system. Differential cordings (SV-ST) were also made in some cases.
Fig. 1. (a) Waveform of the stimulus. Sinusoidally enveloped pip of 98.8 kHz or 143.5 kHz was selected to minimize undesirable transients. (b) Electrodynamic ultrasonic transducer used in this study had a resonant frequency of 98.8 kHz and resonant impedance of 590 52. (Scale in centimeters.)
The intensity of the ultrasonic stimulus was taken to be the peak-to-peak voltage of the signal at the amplifier output to the transducer, using a maximum peak-to-peak voltage of 20 V as the reference (0 dB). In this case, the delay time was negligible. The ultrasonic transducer, shown in Fig. lb, had a lowest resonant frequency of 98.8 kHz and a resonant impedance of 590 0. Another transducer resonating at 143.5 kHz was also used in several cases. Cochlear potentials were recorded through a silver-wire electrode placed on the ventral edge of the round window, with reference and ground electrodes being located on the denuded neck muscles. In some animals, differential recordings were obtained by inserting needle electrodes through small burr holes into the Scala vestibuli (SV) and the Scala tympani (ST) of the basal and second turns of the cochlea [6]. The responses were amplified with a Grass P-18 amplifier and then averaged 32 times with a signal processor (San-Ei, 7T07A). A diagram of the experimental system is shown in Fig. 2. The responses evoked by clicks and ultrasound
re-
were recorded in 12 control animals, and also in animals treated with ethacrynic acid, kanamycin, or hypothermia. A single dose of 40-50 mg/kg of ethacrynic acid (Edecrin; Merck, Sharp & Dohme) was given through an indwelling catheter in a neck vein to each of four animals. Nine animals were made kanamycin toxic by subcutaneous injection of kanamycin sulfate (Meiji, 450 mg/kg) daily for a week, 14-46 days before testing. The ECG was monitored throughout the experiment, and body temperature was monitored by a rectal probe and maintained between 36.5 and 38.5”C except for the, hypothermia portion of the study. Results Normal response
In 12 animals clear, reproducible responses were obtained to ultrasonic stimulation. The ultrasonic-evoked response (UER) waveform was composed of an initial positive deflection, followed by two or three sharp negative peaks (Fig. 3a), and closely resembled the click-evoked responses (CER) shown in Fig. 3b. When the stimulus intensity was lowered the amplitude of the UER decreased and finally disappeared at about - 20 to - 30 dB (2.0-0.6 V peakto-peak) (Fig. 4). The mean value of the stimulus intensities for the minimal detectable level was 1.71 V peak-to-peak (- 21.4 dB). When the stimulus intensity was reduced the latency of the first negative peak increased slightly perhaps as the result of a delay in the time to reach effective stimulus level [4].
145
trigger .
> 0’ 0 m
Frg. 3. (a) The ultrasonic-evoked response (UER) is composed of initial positive and succeeding negative deflections. They are identified to be SP and AP, respectively. SP amplitude is measured from the baseline to the initial peak, and AP amplitude is measured from the initial peak to the first negative dip. (b) The click-evoked response (CER) from the same ear. UER and CER resemble each other except in the prominent SP of the former.
The interstimulus interval (ISI) was found to influence the UER waveform. By decreasing the ISI from 200 to 2.5 ms, the amplitude of the first negative peak was reduced by 50 or 60% but no change was noted in the initial positive deflection (Fig. 5). Asphyxic anoxia, induced by stopping the ventilator, produced substantial changes in the UER waveform. However, the recovery process began immediately after the restoration of artificial ventilation (Fig. 6). The time courses of the amplitudes of the two components of the response seen after 2 min of asphyxia are shown in Fig. 7a and b. Considering the physiology of the cochlea, these negative peaks with adaptation phenomena are probably cochlear action potentials (AP). In addition, differential electrodes in the basal turn of the cochlea detected another form of response. With the onset of asphyxia this potential changed in much the same way as did the initial positive deflection of the
a 0
-- 6.0
-16.5
+* 1 -22.5
v
(dB r~
20V~)
d’
l0i
_ [ees
mt
HII]
I
50
-40
30
-20
IQter7,sl
Fig. 4. (a) Strmulus attenuation causes a decrease in UER amplitude. and UER disappears at about input&output functions of the AP in the UER (- - - - -) and CER ( -). The curves of the UER are the CER. (Responses from the same ear are plotted with the same symbols.)
10
ti,
!c1B!
~20
to -30
far
steeper
than
0
dB. (b) The are
those
of
100
50
25
goxl----20
10 t-4
2 ms
5
>s
H
(m set)
Fig. 6. UER changes induced by 2 min asphyxia and afterward. Complete recovery takes place about 7 mm after resumption of respiration (120 t).
2 mssc Fig. 5. Effect of reducing the interstimulus interval (ISI) upon the UER. The initial positive component shows no particular change, but the following negative components show remarkable adaptation which is considered to be neural in origin.
UER recorded at the round window, although those two responses were opposite in polarity (Fig. 7~). No response component comparable to that from the basal turn was detected from the second turn of the cochlea (Fig. 8). It may be concluded that the component of the response from the basal turn is the summating potential (SP). Direct observation of the differentially recorded responses from the basal turn revealed a microphonic-like component with an amplitude of about 200 PV (peak-to-peak) to be overlapping the SP. This component had the same frequency as the stimulus signal and exhibited remarkable lability in response to anoxia (Fig. 9). Thus it was identified as a cochlear microphonic potential (CM). No
CM was recorded cochlea.
from
the second
turn
of the
Effect of ethacrynic acid The UER were recorded from four guinea pigs before and after intravenous injection of ethacrynic acid. In the first animal, 50 mg/kg of ethacrynate reduced the SP component of the UER to zero within 9 min, inverted the polarity of the response between 9 and 11 min, and caused it to reach a low point at about 11 rnin after administration. Then a slow recovery process occurred and the SP, after a transient overshoot, returned to the original level. The second animal was given 40 mg/kg of ethacrynate, and SP and AP amplitudes were reduced to 40% and 608, respectively, of the preadministration values within 25 min of administration. In the third animal, administration of 50 mg/kg ethacrynic acid led to rapid disappearance of potentials; no recovery of AP was noted within
147
AP
is-
---
-ie
Fig. 7. Effects of asphyxia on amplitudes of AP (a). SP (b), and DIF-SP (c) from the basal turn of the guinea pig cochlea. Closed triangle (A) marks resumption of respiration; the transient overshoot about 446 min after the onset of asphyxia is characteristic.
a
b
Fig. 8. Representative traces from the basal turn (a) and from the second turn (b) of the guinea pig cochlea showing differen-
Fig. 9. Direct observation of differentially recorded potentials. An ultrasonic signal of 98.8 kHz was used as the stimulus (upper trace). The CM response contained overlapping AP and SP (middle trace). There was an obvious reduction of CM amplitude after 2 min of asphyxia (bottom trace).
4 h. A similar effect was observed in the last animal, in which the differentially recorded SP (SV-ST) disappeared rapidly. The effects of ethacrynic acid on SP and AP amplitudes are shown in Fig. 10. In general, administration of high doses of ethacrynate produced a rapid depression of AP and SP within a few minutes, followed by a recovery process which took several hours. This phenomenon is quite similar to the effect of ethacrynate upon the acoustically evoked AP and SP [2]. Effect of hypothermia In three animals the effect of body temperature upon the CER and the UER was examined, using maximal stimulus levels. For both responses, hypothermia led to decreased amplitude, increased latency, and broadening of the waveform, as shown in Fig. Il. Both responses were affected by hypothermia in much the same way. Other authors have investigated the effect of body temperature on AP latency, and determined that the main site of this phenomenon is the afferent hair cell-neuron synapse [15]. The results tially recorded SPs and how they altered during and after 2 min of asphyxia. No significant SP was detectable in the response from the second turn.
-iBo--*
240
2
SP __ __.___-.--------A---v__
120
160
240
4
Fig. 10. Ethacrynate caused SP and AP to decrease rapidly in four guinea pigs (1,2,3,4) within a few min after injection. SP recovered more rapidly than did AP.
of the present tion does not any other site cells just like sounds.
study suggest that ultrasonic perceptake place at the cochlear nerve, nor central to it, but in the cochlear hair the perception of normal, audible
Responses in the kanamycin-treated animals Kanamycin
(KM) treatment
reduced
the ampli-
tude of the UER and increased AP latency IO varying degrees. The mean value of the amplitude of the response to maximum ultrasonic stimulation was 127.1 pV, which was about a quarter of the normal value (560.4 pV). while AP latency at maximum stimulation was 1.21 f 0.14 ms, significantly greater than normal (0.95 + 0.06 ms). This difference in latency remained almost constant at all stimulus levels (Fig. 12). Discussion Although ultrasonic perception (USP) in man is not always well known even to audiologists or physiologists, its existence is now beyond doubt. However, lack of appropriate methodology to investigate this phenomenon objectively has precluded the conducting of a detailed physiological study of the mechanism by which this occurs. Thus, it seems extremely important to develop an animal model so that this subject can be investigated more fully. Behavioral studies have shown that the uppermost frequency of the guinea pig auditory field lies around 50 kHz [9]. The signal frequencies used in the present experiment (98.8 and 143.5 kHz) are, therefore, obviously ‘ultrasonic’ to the guinea pig. Nevertheless, the results of the present study showed that such signals evoke cochlear potentials just as do sounds within the auditory field. This is considered to be objective evidence that guinea pigs have ultrasonic perception. Although several hypotheses have been proposed to explain the perception of ultrasonic stimuli (Table I, [S]), the mechanism by which such perception occurs still has not been determined. In the present study, ultrasonic stimuli resulted in clear SP and CM, in addition to AP. Furthermore, these potentials were affected by anoxia, hypothermia, and the administration of kanamycin or ethacrynic acid. These results strongly suggest that the SP and CM recorded in the present experiment were not artifacts but rather evoked by ultrasound. Therefore, it is reasonable to presume that the primary site of ultrasonic action may be in sensory hair cells of the basal turn of the cochlea rather than in the cochlear nerve. In a recent report [13], AP evoked by ultra-
149
C
b UER
CER 2
Fig. Il. (a) Representative traces of UER and CER during body cooling (hypothermia). Amplitude decrease, latency increase, and decreased sharpness of AP are evident in both the UER and CER. The effect of body temperature on AP latency of UER is shown in b. and of body temperature on AP latency of CER in c; there was no significant difference between these responses.
sound were compared with those evoked by clicks. The former were found to have a steeper input-output function and poorer adaptation to a
reduction in ISI than the latter. These results indicated that auditory potentials evoked by ultrasound are similar to those evoked electrically by
b
a
. -28
I
-Ifi
Intensity
[dBl*
direct stimulation of the cochlear nerve. However, the results obtained in the present study, especially the presence of CM and SP evoked by ultrasonic stimuli and the alterations in AP
I
HYPOTHESES
ON ULTRASONIC
PERCEPTION
I. Utrasonic perception as a result of inadequate stimulation particular sections of the hearing system: (1) Direct stimulation of co&ear nerve fibers. (2) Stimulation of organ of Corti in some different manner.
of
2. Ultrasonic perception as a result of adequate stimulation of the inner ear: (1) The existence of rudimentary ultrasonic receptors. (2) The generation of subharmonic vibrations in the range of audible frequencies through non-linearities in the transmission path of ultrasonics. (After
Dieroff
and Ertel [8], partly
modified.)
-ia
Intensity
Fig. 12. (a) AP input-output UER functions of normal (-; Kanamycin treatment reduces the AP amplitude to about a fourth kanamycin-treated animals. AP latency is considerably elongated
TABLE
--i&3
idR1°
n = 12) and kanamycin-treated (- - - - - -; n = 9) animals. of normal. (b) AP intensity-latency UER functions of normal and in the latter. (Vertical bars indicate standard error of the mean.)
latency noted with hypothermia, permit us to discard the cochlear nerve stimulation theory. This theory may be called the ‘inadequate stimulation’ hypothesis. It is also evident that the subharmonits theory, according to which the sound heard during exposure to ultrasonic stimuli is merely a distortion product within the auditory field, is not adequate to explain this phenomenon. Deatherage et al. observed that the pitch of the sound heard when ultrasonic stimulation is presented by bone conduction is the highest pitch which a subject can hear [7]. Then Dieroff and Ertel showed that ultrasonic stimuli are always determined to be in the frequency range 13-16 kHz, independent of the stimulus frequency (20-100 kHz) [8]. This shows the poor frequency resolution of this sensation. In addition, we previously reported upon the extremely prominent maskability of a bone-conducted ultrasonic signal
151
upon an audible sound above 5 kHz [12]. Such masking is clearly an exception to the general rule, are not effective at masking that is. “maskers lower frequency probe tones”. The most appropriate explanation for this latter phenomenon seems to be that the ultrasonic vibration stimulates the basal portion of the organ of Corti just enough to permit perception of a higher-frequency audible sound. This concept is not particularly new. In 1954 Deatherage et al. [7] described a similar mechanism which they called ‘diplacusis’, by which direct stimulation of the basal end of the cochlea led to perception of a sound. Data on the absolute thresholds for sonic and ultrasonic bone-conducted frequencies measured in human subjects by Corso [5] also support such a mechanism of inadequate stimulation. In the latter study an absolute threshold curve was obtained which showed a very marked rise (about 50 dB/octave) in the 14-20 kHz region, and a far more moderate slope in the ultrasonic region. This led to speculation that there may be a difference in the way sonic and the ultrasonic stimuli are perceived in the cochlea. It is impossible to conclude from the results of the present study whether ultrasound directly stimulates the hair cells or whether the basilar membrane is caused to vibrate first. As shown in Fig. 4b, the input-output curve of the AP evoked by ultrasound is steeper than that evoked by an audible sound. One possible explanation for this difference may be that the guinea pig ear normally recruits in these high-frequency regions. Recruitment of the whole-nerve AP input-output function, as may be found in some pathological ears, is attributed to loss of the tip of the single-unit tuning curve at the characteristic frequency (CF) [14]. If it is assumed that the sharp tuning mechanism does not function in ultrasonic perception, this difference can be explained easily. The results of the present study suggest that the mechanism by which hair cells are stimulated by ultrasound is different from that by which they are stimulated by audible sound. Thus, ultrasonic perception, at least in guinea pigs, is due to the activation of hair cells in the basal turn of the cochlea in quite a different manner from the way
in which they are activated the auditory field. Further clarify this phenomenon.
by normal sound within studies are necessary to
References 1 Abramovich, S.J. (1978): Auditory perception of ultrasound in patients with sensorineural and conductive hearing loss. J. Laryngol. Otol. 92, 861-867. 2 Aran, J.-M. and Charlet de Sauvage. R. (1977): Evolution of CM. SP and AP during ethacrynic acid intoxication in the guinea pig. Acta Otolaryngol. 83. 1533159. 3 Bellucci, R.J. and Schneider, D.E. (1962): Some observations on ultrasonic perception in man. Ann. Otol. Rhinol. Laryngol. 71, 719-726. 4 Charlet de Sauvage. R., Cazals, Y.. Erre. J.-P. and Aran. J.-M. (1983): Acoustically derived auditory nerve action potential evoked by electrical stimulation: An estimation of the waveform of single unit contribution. J. Acoust. Sot. Am. 73, 616-627. 5 Corso. J.F. (1963): Bone-conduction thresholds for sonic and ultrasonic frequencies. J. Acoust. Sot. Am. 35. 173881743. 6 Dallas. P.. Schoeny, Z.G. and Cheatham. M.A. (1972): Cochlear summating potentials: descriptive aspects. Acta Otolaryngol. Suppl. 302, 1-46. 7 Deatherage. B.H., Jeffress, L.A. and Blodgett. H.C. (1954): A note on the audibility of intense ultrasonic sound. J. Acoust. Sot. Am. 26, 582. 8 Dieroff. H.G. and Ertel, H. (1975): Some thoughts on the perception of ultrasonics by man. Arch. Otorhinolaryngol. 209, 277-290. 9 Heffner, R., Heffner, H. and Masterton. B. (1971): Behavioral measurements of absolute and frequency-difference thresholds in guinea pig. J. Acoust. Sot. Am. 49. 1888-1895. im Ultraschall10 Kietz. H. (1951): Hiirschwellenmessung gebiet. Acta Otolaryngol. 39. 183-187. 11 Kunze, W. and Kietz. H. (1949): Uber Horempfindungen im Ultraschallgebiet bei Knochenleitung. Arch. Ohr. Nas. Kehlkopfheilkd. 155, 6833692. K. and 12 Ohyama, K., Kusakari. J., Takasaka, T., Kawamoto. Yuasa. R. (1980): Investigation of human ultrasonic percepnon. Audiology (Japan) 23, 297. 13 Ohyama, K.. Kusakari, J., Takasaka, T., Kawamoto, K. and Yuasa. R. (1982): Cochlear and brainstem response evoked by ultrasonic vibration. Audiology (Japan) 25, 79-83. 14 Gzdamar, 0. and Dallas, P. (1976): Input-output functions of cochlear whole-nerve action potentials: Interpretation in terms of one population of neurons. J. Acoust. Sot. Am. 59, 143-146. auditory adaptation. II. 15 Prijs, V.F. (1980): On peripheral Companson of electrically and acoustically evoked action potentials in the guinea pig. Acustica 45. l-13. 16 Pumphrey, R.J. (1950): Upper limit of frequency for human hearmg. Nature (London) 166, 571.