Development of the cat peripheral auditory system: input-output functions of cochlear potentials

Brain Research, 219 (1981) 29-44

29

Elsevier/North-Holland Biomedical Press

DEVELOPMENT OF THE CAT PERIPHERAL AUDITORY SYSTEM: I NP UT- OUT PU T FUNCTIONS OF COCHLEAR POTENTIALS

D A V I D R. M O O R E

University Laboratory of Physiology, Parks Road, Oxford OX1 3PT (U.K.) (Accepted January 1st, 1981)

Key words: cat - - auditory system - - cochlea - - development

SUMMARY

Compound auditory nerve action potentials (APs) and cochlear microphonics (CMs) were recorded from the round-window of kittens aged 3-9 weeks and of adult cats. Animals were anaesthetized and pure tone stimuli were delivered via calibrated, sealed, transducer systems. AP and CM amplitude and AP latency were measured over a wide range of stimulus intensities (up to 80 dB SPL) and at 5 octave-interval stimulus frequencies (1-16 kHz). At low stimulus intensity levels, AP amplitude had attained adult levels to low and high frequency stimuli by 6½ weeks of age and to mid-frequency stimuli by 9 weeks. As stimulus intensity levels were increased, the kitten input-output functions diverged progressively from those of the adults. At these higher intensity levels, AP amplitude maturation in even the 9 week animals was incomplete. AP latencies to stimuli of all frequencies shortened between the third and fourth weeks but remained stable thereafter. CM amplitude also reached maturity by the fourth week. These findings suggest that the development of AP after the fourth week consists of an increase in the synchrony of auditory nerve fibre responses, since both the fine structure of the cochlea and the responses of single nerve fibres are known to be mature by the end of the first postnatal month.

INTRODUCTION

The introduction of electrophysiological procedures into clinical audiologyl~,44, 48 has, over the last few years, resulted in a renewed interest in the properties of cochlear potentials recorded with 'macro' electrodes. In this context the development of cochlear function is of particular interest, as traditional methods for assessing human auditory abnormalities (e.g. B6k6sy audiometry) are based on behavioural responses that are inapplicable to the examination of infants. Several recent studies ~7, 0006-8993/81/0000-0000/$2.50 © Elsevier/North-Holland Biomedical Press

30 45,46 have reported the development of scalp-recorded auditory brain stem-evoked responses (BERs) in humans and have shown that latency of all BER components, including those attributed to the cochlea and auditory nerve, decreases as a function of age. There are, however, two problems with these studies. The first is one inherent in all BER work, whether performed on humans or animals tT,at,~7 ,47, namely the position of the source of origin of the potentials. The second problem is a methodological one; stimuli have consisted of clicks of mixed spectral content and poorly specified intensity. It is well known that response latency of the auditory nerve compound action potential (AP) varies markedly with both stimulus frequency and intensity s. It is possible to overcome these problems by studying cochlear potentials using electrodes situated either inside, or immediately adjacent to the cochlea and by using pure-tone stimuli of known intensity. Investigations of cochlear potential development have been performed in the mouse 2,26, rat 7, gerbil 11, guinea pig35,ag,41,42, rabbit a and cataa,as,39,42, 4a. These studies have been mainly concerned with the onset time (i.e. earliest age at which a response can be recorded), effective stimulus frequency range, threshold and latency of both AP and the cochlear microphonic (CM). In all species examined, the onset of CM occurs a few days after birth with AP onset following some 2-3 days later. By the above indices, CM and AP remain immature until between 15 and 30 days after birth, depending on the species and index chosen. This timing is coincident with the final anatomical development of the cochlea 34,35,37. However, the basilar membrane is not a uniform structure along its length. Low frequency tones maximally displace the apical end of the membrane and high frequency tones only cause displacement of the basal end. Since the cochlea is known33,36, 37 to mature anatomically from the base towards the apex, it might be predicted that cochlear potential responses to high frequency tones would be fully developed before responses to low frequency tones. In an attempt to study the frequency selectivity of the developing cochlea some authors have used cochlear potential 'audiograms'. These are plots of an arbitrary response criterion as a function of stimulus frequency. In the opossum 24,~5 and the rat 7, round-window recorded CM responses are initially restricted to stimuli of low frequency. The effective frequency range then gradually broadens with age to include successively higher frequencies. In the gerbiP 1, on the other hand, CM responses in a 12 day animal were restricted to high-frequency stimuli. By 16 days the effective frequency range had broadened dramatically, but the audiogram retained the greatest elevation (relative to a 90 day animal) in the low frequency end of the spectrum at all ages. Although there is a discrepancy between the results of these studies which may be due to species specificities, neither of the results is incompatible with the anatomical evidence outlined above. This is because round-window recorded CM is not a good measure of the functional development of different regions along the cochlear partition. CM is known to originate in the hair cells 9,49,51 and round-window recorded CM represents the sum of the outputs of many out-of-phase generators at different distances from the round window. An audiogram derived from these measures will therefore largely reflect the frequency sensitivity of a subset of hair cells, namely those located in the basal end of the cochlea.

31 An alternative approach is to study AP frequency selectivity. Behavioural audiograms have been shown to be correlated with the envelope of cochlear nerve unit thresholdslS, ~a and AP audiograms correlate highly with mean inferior colliculus unit thresholdsZL Since unit thresholds are similar at all levels of the primary auditory pathway, it is reasonable to assume that AP audiograms have a good correlation with behavioural audiograms. No such correlation has been established for CM, and a comparison of rat CM 7 and behavioura114 audiograms suggests that such a correlation would be poor. Moore and Irvine 29 presented AP audiograms for both kittens (11-55 days) and adult cats. AP 'thresholds' (20 #V citerion) were elevated (in adults) over a wide frequency range in all kittens up to 35 days of age. In older animals threshold elevation was limited to frequencies above 3 kHz and a slight mid- to high-frequency elevation was still apparent at 50 days. While these findings appear to be at variance with the anatomical evidence cited above, single unit thresholds in both the cochlear nucleus (CN) 4 and inferior colliculus (IC)1, 29,53 attain adult levels at an age that is comparable (20-30 days in cats) to the time of anatomical maturation of the cochlea. In the study reported here, an attempt was made to clarify the nature of these discrepancies by examining AP development as a function of both stimulus frequency and intensity. MATERIALS AND METHODS Subjects

Data were obtained from 5 adult cats and 15 kittens. The kittens were aged between 20 and 68 days and were arranged into 4 age groups according to Table I. Kittens were raised in the Monash University breeding colony and, at the time of testing, had open, well-formed ear canals and middle ears that were clear of any fluid or mesenchymal tissue. Adult cats weighed 2.5-2.9 kg. Although the precise age of these cats was unknown, previous observations 2s suggest that this weight range is indicative of an age of at least 5 months. All animals were in good health. Stimulation

Pure tones were generated by a Rockland 5100 digitally controlled frequency synthesizer, shaped by an electronic switch (50 msec duration, 1 msec rise/fall time) TABLE I Age grouping o f cats Group designation (weeks)

Age range (days) Mean age of group (days) Number of cats

3

4

6.5

9

Adult

20-22 21 3

27 27 2

39-51 45 6

58-68 63 4

Adult -5

32 and presented at a rate of 5/sec. Click stimuli were produced by transient, squarewave pulses (0.1 msec duration) applied to the transducers. The stimuli were transduced by a 0.5 in. condenser microphone (Briiel and Kjaer (B&K) Type 4133) mounted in a stainless-steel speculum-coupler. This assembly also housed a recording microphone (B & K, Type 4134) coupled to a probe tube. Two sizes z9 of speculumprobe tube assemblies were used as required to fit different sized ear canals. Supplementary sealing of the speculum, using Vaseline, was also occasionally used. Calibration of each assembly was carried out, in situ, at the commencement of every experiment and in the manner described elsewhere 1~. Following calibration, the cavity correction factors were printed out and these were used to set sound pressure levels (SPLs) during the experiment, by means of digitally controlled attenuators (Grason Stadler 1284). All SPLs are expressed in dB (2 × 10-5 N/sq.m).

Procedure Young kittens were anaesthetized with ketamine hydrochloride (Ketalar, 35 mg/kg i.m.) followed, some 20 min later, by sodium pentobarbital (Nembutal, 15 mg/kg i.p.). Adult cats and older kittens were anaesthetized with Nembutal (35 mg/kg). Supplementary doses of anaesthetic were administered whenever the animal showed a reflex withdrawal to a pinch of the forepaw. Rectal temperatures were monitored throughout and maintained at 37.5 °C (adult) or 37 °C (kittens). Following tracheotomy and cannulation, the dorsal surface of the skull was cleared bilaterally down to the level of the external auditory meatus. The bullae were also cleared of overlying tissue and the meatuses were transected to enable placement of the stimulus assemblies to within 1-2 m m of the eardrum. Steel screws were fixed in the skull and the head was cemented to a modified Trent-Wells stereotaxic frame. A small hole was made in the bulla by means of the pointed tip of a scalpel blade and a stainless-steel spring electrode, insulated to within a few millimeters of the tip, was placed on the round window. Preliminary observations of AP amplitude in the adult cat showed no marked difference between an open bulla preparation and one in which the electrode was sealed into the middle ear using dental acrylic. All data reported below were obtained from open bullae. Cochlear potentials were amplified and filtered (80 Hz high-pass) by two Tektronix Type 122 preamplifiers, connected in series to give an overall amplification of 104-105 , and displayed on an oscilloscope. The reference electrode was fixed to a saline-soaked ball of cotton wool which was embedded in the neck musculature. For AP analysis the output of the preamplifiers was processed by a Data General Nova Computer programmed for signal averaging. The averaged response was displayed on an oscilloscope and selected response parameters (see below) were printed out on a Teletype. Tone burst stimuli were of rolling phase. By averaging a large number of stimuli, the CM component of the response could be eliminated, leaving the AP intact. This procedure enabled measurements to be made on AP alone. In the case of clicks, CM could be averaged out by reversing the polarity of the square wave generating pulse after half of the stimuli had been presented. All AP data are based on a sample of 200 stimuli, an analysis time of 20 msec and a sampling bin width of 0.1

33

msec. AP amplitudes were defined as the peak-to-peak response of the first biphasic wave of the AP. AP latency was the time from stimulus onset to first negative minimum (i.e. N1 component). Each of these parameters was read off digitally being, respectively, the difference between the maximum and minimum bin counts during the sampling period (except in those few cases when N2 was larger than N1), and the bin in which the minimum count occurred. CM amplitude was measured directly from the oscilloscope, without averaging. The minimum CM amplitude that could be measured by this method was 30/~V. Both ears were tested, in all but 7 animals, and the responses of the two ears of each animal were averaged to produce a single datum for each intensity/frequency combination. In the remaining cases, data points consisted of the responses of a single ear. Click stimuli were tested first, followed by 11 pure-tone frequencies. The maximum intensity used, at any frequency, was 80 dB. Data were collected at each successive 5 dB attenuation from 80 dB, until an AP of less than 3/~V was obtained. For clarity of presentation, only the data for 5 frequencies, at octave intervals from 1 to 16 kHz, are presented here. Some additional kitten data, obtained at 30 kHz, are also presented. The remaining data were collected at frequencies intermediate to the octave intervals and the trends in these data were consistent with those presented below. RESULTS

General features of averaged AP responses E x a m p l e s o f a v e r a g e d A P r e s p o n s e s f r o m a d u l t c a t s a r e s h o w n i n Fig. 1. T h e

A

B

C

Ii

D

i t

E

F

G

H

I

J

K

L

I~1

N

[

i

0

l 6 msec

Fig. 1. Examples of averaged cochlear potentials from adult cats. A - D : cat MP-05, right ear. A: 12 kHz tone, 40 dB. B: 12 kHz, 50 dB. C: 2 kHz, 60 dB. D: 2 kHz, 65 dB. E-I: cat MP-07, right ear, 16 kHz. E: 40 dB. F" 50 dB. G : 60 dB. H: 70 dB. I : 80 dB. J - O : cat MP-07, right ear, click. J: 30 dB. K : 40 dB. L: 50 dB. M: 60 dB. N: 70 dB. O: 80 dB. Amplitude calibration: 20/~V.

34 120

100 1 kHz

;

60 2kHz

4kHz

/

)J

,I

~60 iii

~

/

.'C

,

, ~' ? ~

(3

i ,1(30 1Q 210

; ~ / ~' ' ~ z ~,

..~ n "5" 2( (3_ <

6o

_ ,.o 3'o '0

t

~'o 60 ~o 20

. . . . . 20 30 ~.0 16 kHz

310 4;

L 60

slo

i 70

50

60

# 0

810

8 kHz

80

.....

i

/'

' //LJ /

' 80 6O ~

/ / p''

Y

~'

30 kHz

Y ~/

20

0

0

310

20

510 610 710 810

20 310 /.10 SO 610 710 810 I N T E N S I T Y (dB)

10

20

310

/,'0

Sl0

60

710 S-~0

Fig. 2. AP input-output functions. The horizontal, broken lines are at 3 HV and represent the minimum amplitude APs recorded. Each data point is the mean from at least 4 ears. Symbols : filled

circles, 3 week group; open circles, 4 week group; filled triangles, 6.5 week group; open triangles, 9 week group; open squares, adult. degree of synchronization of the AP response was a function of stimulus frequency. At frequencies higher than 4 kHz (e.g. 12 kHz; Fig. 1A, B) APs had short latencies, abrupt rise times and short overall durations compared with responses to lower frequency stimuli (e.g. 2 kHz; Fig. 1C, D). It is likely that the rolling phase averaging procedure contributed to the poorer synchronization of the low frequency responses. That this factor was not a sole determinant of low frequency desynchronization is suggested by the difference between the duration of low frequency-evoked N1 and the period of the stimulus (e.g. 2.5 msec Nt vs 0.5 msec for a 2 k H z stimulus, Fig. 1C, D). Another feature of high frequency responses was the presence of Nz components. Although N2 was not studied systematically, thresholds were higher than those of N1 (Fig. 1E-I) and, in some cases, Nz amplitudes at 70-80 dB stimulus level were higher than those of N1. Summating potentials (Fig. 1H - l ) were obtained at all stimulus frequencies, but only in response to relatively high stimulus intensities. These potentials were filtered out before measurements of AP amplitude were obtained. Superimposed on each of the above responses was an increase in N1 amplitude with stimulus intensity. This feature was seen most clearly when click stimuli were

35 employed and an example of intensity-related changes in click-evoked APs is shown in Fig. 1J-O. A final point concerning the general characteristics of APs is that, in a similar fashion to the adult cats, the features described above were seen in all age groups of kittens.

AP input-output )'unctions Complete AP input-output functions for 6 pure-tone stimulus frequencies are presented in Fig. 2. For 1 k H z stimuli, 3 #V APs could not be obtained from adult cats at intensities less than 35 dB. Above this level AP amplitude increased, slowly at first, and then more rapidly as the stimulus level exceeded 55 dB. This curvilinear increase of amplitude with stimulus intensity was seen in adult functions at all frequencies and this trend was also apparent in the kitten groups. Little evidence of the classic two stage AP input-output function6, s is apparent in the adult curves of Fig. 2, but it is possible that the stimulus intensity levels used in the present study were too low to activate the mechanism responsible for the high-threshold portions of the classic function. At low ( < 30 dB) stimulus intensity levels, AP amplitudes from the two eldest kitten groups were similar to those of the adult group at low and high frequencies. In the mid-frequency range (4 and 8 kHz) some residual elevation was still present in the 6½ week group, but this had disappeared by the ninth week of life. In contrast, the intensity required to produce a 3 y V AP in the 3 week group was markedly higher than in the adults. As stimulus intensity was increased the input-output functions of the kitten groups diverged progressively from those of the adults. The degree of this divergence varied with frequency. At 1 k H z the adult function rose very rapidly from 50 dB, while none of the kitten functions showed such a dramatic increase in amplitude. At 2-8 kHz the functions for all kitten groups diverged from the adult function at a rate that was inversely proportional to the age of the group. At 16 kHz,

8o

A 3pV

12o

B. 20pV

~\\

,oo

\/~

.o

o

FREQUENCY (kHz)

Fig. 3. AP audiograms. A: 3/zV criterion. B: 20 yV criterion. Filled squares in A are mean adult inferior colliculus unit thresholds, within each octave band, reported elsewhere2L Other symbols represent the age groups and are equivalent to those shown in Fig. 2. Intensity levels above 80 dB in B (broken line) were derived from linear extrapolation of the relevant AP input-output functions.

36 A. 3

pV

B. 2 0 N V

.80 *70

~- -50

.60

~ +50 O ~ 40

*50 *z.0 "o

F- .30

/ //

"-\

.30 // / /

,~-20

5 ~-_oF / / ~r

~

"" \

..... ~

.10 0

~

& ul

2

&

8

o

lo

16

FREQUENCY

1

2

&

8

(kHz)

Fig. 4. Elevation of kitten AP audiograms relative to those of the adult cats. A : 3/~V audiograms. B : 20/zV audiograms. Age group symbols as in Fig. 2.

the function for the 9 week group was almost identical with the adult function. Moreover, at both 16 and 30 kHz, the functions from the various age groups were more nearly parallel than at lower frequencies. Data bearing directly on the issue, outlined in the introduction, of variation in criteria for AP maturation are shown in Figs. 3 and 4. AP audiograms (Fig. 3) for adult cats had a very similar shape for both a 3 and a 20 #V criterion, with the former following a course at about 20 dB lower intensity than the latter. Mean IC unit thresholds for adult cats, derived from a previous study zg, are indicated by the dashed line in Fig. 3A. It is noteworthy that the 3 # V audiograms for both the adult and eldest kitten group are very close to the mean IC unit threshold curve. For the younger kitten groups 3/zV audiograms have a similar shape to the adult curve, but are somewhat elevated, particularly at 4 and 8 kHz. A rather different picture emerges if the 20 #V audiograms (Fig. 3B) are considered. As has been reported previously zg, 3-week-old kittens show a large audiogram elevation over the entire frequency range examined. In the older kitten groups (4 and 6½ weeks), however, this elevation was restricted to the higher stimulus frequencies. By the ninth week, audiograms were very similar to the adult function, although a slight elevation seemed to remain at 4 kHz. To enable a more direct comparison of audiogram elevation as a function of age, the data of Fig. 3 are replotted in Fig. 4 as AP 'threshold' elevation relative to the adult values. It is clear that the higher AP amplitude criterion (20 #V) results in a much larger mid- to high-frequency elevation of the kitten audiograms than does a 3 /~V criterion. Another point of interest concerning Fig. 4 is that, for 3 #V responses at 16 kHz, the 6.5 and 9 week kittens are about 10 dB more sensitive than the adults. It is possible that this observation is attributable to a loss of high frequency sensitivity in the adult cats. Such a hypothesis is consistent with Liberman's 19 finding of a difference in single auditory nerve fibre sensitivity between cats reared in a low-noise environment (as were the kittens in the present study) and those obtained as adults with an unknown acoustic history.

37 It might be argued that the smaller amplitude, averaged APs found in the kitten groups, were a result of the relatively high stimulus repetition rate (5/see). According to this argument, the developmental trends shown in Figs. 2-4 would reflect a diminution in response amplitude across the 200 stimulus trials rather than an absolute 'one-trial' amplitude decrement. However, observation of AP amplitude in one of the youngest kittens (21 days) showed no rate effects on response amplitude, to an 8 kHz tone, at repetitions of less than 10/see.

AP latency There is considerable controversy concerning which auditory nerve fibre sub-

3,8

d/~/~

3.8 f

3L

34

26

W

20i

a X

.~'-36

\\

,-J -,3,2

/~

40 (SO s6r

6.s

y

I 3.2

2.8

~

26

.80 \

1/.

"

I\Y i

I 60

i

20

~o

y~ 9

[ 3.2

2,8

~c 60

"~AD ULT "

28

2.0

1.6

1.2

I

20

I

I

~o

I

I

60

I

I

80

"

I

20

I

I

~o

I

6'o

I

8'o

"

2'o

I

2o ' 6'o ' 8'o

INTENSITY (dB) Fig. 5. Latency from the beginning of the stimulus rise time to the first minimum (N1) of the compound AP. Each pair of co-ordinates contains the data for the age group indicated above the function. Smaller numbers to the right of each function indicate the stimulus frequency(in kHz). Open squares, click (C) functions.

38

~3 39

Q

3 5

~

27

W23

.25 o,;0

~o

•60 -80

r

FREQUENCY

i 8

E

(kHz)

Fig. 6. AP latency-stimulus frequency functions (9 week group) at each of 4 intensity levels (25, 40, 60 and 80 dB). The 1 and 2 kHz points, joined by the broken lines, in the 25 dB curve were derived from linear extrapolation of the latency functions in Fig. 5. The unlabelled, broken-line curve is the cochlear travel time function for the adult cat (adapted from Gibson et al.la). population contributes to the compound AP. Some evidence 1s,5°, derived from a comparison of AP with single unit activity in the auditory nerve or cochlear nucleus, suggests that the basal end of the cochlea is principally responsibe for N1, whatever the stimulus frequency. However, more recent studies31, 32 have argued that pure-tone generated AP reflects the activity of fibres having the same characteristic frequency as the carrier frequency of the tone burst. 0 z d a m a r and Dallos 3z have interpreted AP input-output functions as reflecting the summed response of all single auditory nerve fibres excited by a tone of any given frequency and intensity. One aid to identification of the fibre population contributing to a given AP component is the response latency of that component. Latency may also be useful as a means for studying the development of acoustic transmission in the middle and inner ear. N1 latencies for each developmental group are shown in Fig. 5. If it is assumed that transmission times, from the stimulus transducer to any given point on the basilar membrane, are invariant across stimulus frequencies and that peak N1 is associated with the region of maximum basilar membrane displacement, it is apparent from Fig. 5 that each frequency was producing maximal displacement of a different region of the basilar membrane. It is also apparent that low-frequency stimuli were associated with longer latencies than were stimuli of high frequency. These two relationships hold for all developmental groups. An obvious developmental trend in Fig. 5 is the shortening of latencies to stimuli of all frequencies between the third and fourth week. One other difference between groups is the presence, in some high frequency and click-generated kitten functions (3, 4 and 9 week click functions; 6.5 week 4-16 kHz functions), of an asymptotic decline in latency with increasing intensity. N o such asymptotes are apparent in the adult data. A final point of interest is that, in kitten groups 4, 6.5 and 9 weeks, the 1 and 2 kHz functions overlap at many points, particularly at high intensities. No such overlap is present in the adult low frequency functions. The 4 and

39 8 kHz functions also showed some overlap in the 4 week group. With these exceptions, it would appear that AP latency functions are adult-like by the fourth week of life. Perhaps the most direct way to examine the source of N1 is to compare latencies with the travel time of the wave of displacement on the basilar membrane. Estimates of travel time have been derived by Gibson et al. la on the basis of the relationship between the phase angle of discharge and stimulus frequency for phase-sensitive neurones in the adult cat CN. A distance-frequency function15 fitted to these unit data has been reproduced in Fig. 6. Also shown in Fig. 6 are latency data obtained from 9week-old animals in the present study. At low stimulus intensities the latency data are very nearly parallel to the travel time function. For a 25 dB stimulus there is approximately 1.8 msec between the two functions. This represents an acoustic delay (transducer to eardrum), a middle ear delay and a neural delay and is similar to estimates of these delays made by Brugge et al. 4. As intensitiy increased, the variation in latency with frequency decreased. This indicates a breakdown of the good relationship between stimulus frequency and point of maximum basilar membrane displacement found at lower intensities. It may be noted from Figs. 5 and 6, however, that this breakdown was never complete (at least up to 80 dB) and that even at the highest intensity used in this study, low frequency stimulation resulted in maximal basilar membrane displacement at more apical regions than did high frequency stimulation. Thus it may be stated with some confidence that, for low stimulus intensities (less than about 50 dB), the compound AP recorded at the round-window is

500

5O0[

/

1 kHz

2 kHz

200~

200

500

/// 200

..~ao

I..iJ t:3

30

30L

I..-

I

..J

60

I

I

I

70

30

I

I

80

60

¢1 I 500 4 kHz <.

500

I

I

I

I

I

70

500

8 kHz

16 kHz

200 2 0 0

200

80

/

80

30

'

6'0

I 7O /

80

30 I

I

80

I

I

80

60

I

I

70

INTENSITY

I

30 I

80

(dB)

Fig. 7. C M input-output functions. Age group symbols as in Fig. 2.

I

I

70

#

I

80

40 primarily contributed to by those auditory nerve fibres having maximum sensitivity to the stimulus frequency.

Cochlear microphonics It was argued above that cochlear microphonics recorded at the round-window reflect the activity of hair cells located in the basal end of the cochlea, regardless of stimulus frequency. CM was recorded in the present experiments for two reasons. First, there is a fairly large body of data on CM development but, with the exception of one study 28, CM input-output functions have not previously been examined in young animals. Second, it is the basal end of the cochlea that is of interest in attempting to explain the origin of the high frequency/high intensity retardation of AP development. If CM can be shown to have adult-like input-output functions at ages below 9 weeks, the AP retardation described here must be the result of changes in mechanisms more central than the hair cells. CM input-output functions are presented in Fig. 7. All functions show the characteristic s linear relationship between sound pressure level (in dB) and logamplitude found at low to middle intensity levels. Slopes of the functions are close to unity indicating that the recorded CMs were free of noise or AP contaminants 8. The developmental trend in these data is clear; only CMs from the 3-week-old group showed decreased sensitivity relative to the 9 week group. Functions from the older kitten groups almost perfectly overlay one another for some stimulus frequencies (and clicks) and are rather similar for the others. Although CM data were not collected from adults, the functions shown here for the 3 eldest groups are very similar to adult data obtained by others 5z. In terms of response amplitude to a given stimulus, it seems that CM is well developed by the fourth postnatal week. DISCUSSION

Basis of prolonged AP development The results of these experiments demonstrate that the age of attainment of mature APs varies with the amplitude criterion selected. At low stimulus intensities, small amplitude APs may be evoked across a wide frequency range in the 4-week-old kitten. The intensity required to produce a given low-amplitude AP reaches adult levels by about the sixth week, with a slight residual elevation around the midfrequency range. If a higher AP criterion is selected, the results are rather different. Kitten audiograms remain markedly elevated at mid- to high-frequencies until well after the sixth week, with some evidence for mid-frequency elevation still present at 9 weeks. As the criterion AP amplitude is progressively increased, so the gap between kitten and adult audiograms becomes larger. These data strongly suggest that developmental changes are occurring ill the kitten cochlea as late as 9 weeks postpartum. Light microscopic observations a~,36,37 have clearly shown that gross structures in the basal end of the cochlea reach maturity between the tenth and twelfth day. The apex, however, does not reach structural maturity until about the fifteenth day. The fact that AP input-output functions, even

41 at low stimulus levels, are very immature in 3-week-old animals highlights the inadequacy of light-microscopy as a sole means for determining the functional development of the cochlea. A more recent study 44 of cochlea innervation has employed electron microscopy to examine the development of the hair cell-auditory nerve synapse in the basal turn of the kitten organ of Corti. In this region evidence was obtained for a two stage maturation of the cochlea, culminating, at about the end of the third week, with the final development of synapses on the outer hair cells. Although this study suggests a more prolonged development than the earlier, lightmicroscopic work, it nevertheless pre-empts the most obvious explanation of the present results: namely that proper synaptic transmission at the base of the hair cells is not fully developed until after the second postnatal month. In order to derive other predictions as to what is occurring in the cochlea during the second month of life it is necessary to discuss the composition of the compound APs recorded in the present study. AP is thought to represent the synchronous discharge of a number of auditory nerve fibres. According to an estimate made by Davis 1°, the smallest APs recorded in the present experiments (3 #V) may be the compound response of as many as 30 fibres. For larger responses (eg. 2 0 / t V ) the number of auditory nerve fibres required to fire in synchrony will be much higher. Thus it would seem, on the basis of the results presented above, that the smaller the number of fibres required to fire in synchrony, the more rapid is the apparent development of the AP. Single unit studies have suggested that both the number 4 and pattern 5 of discharges in auditory nerve fibres are adult-like by the beginning of the third postnatal week*. However, the further development of the compound AP in the second month must mean that some aspect of the auditory nerve response is immature beyond 3 weeks. This could be either the number of active units or the across-fibre synchrony of the discharges. The finding that CM response amplitude was mature by the fourth week of life suggests that the number of active hair cells in the basal turn of the cochlea is as great as that in the adult. If, as Pujol et al. 34 have argued, the hair cell-auditory nerve synapse is well developed by the third week, it seems likely that the development of high amplitude APs is dependent on an increased synchrony of nerve fibre response. What the basis of these changes is remains unclear. Several studiesl,4,2a, 44 have suggested that middle ear immaturity contributes to the poor responsiveness of auditory neurones in very young animals. The middle ear is fluid-filled until the end of the first week and mesenchymal debris is still present until at least the end of the second week. Beyond this age little is known. It has been shown aa, however, that 'internal growth' of the dog middle ear ossicles may persist for as long as one year postnatally. It is conceivable that a progressive hardening of the ossicles might lead to an increase in the dynamic range of the compound AP. This could result from a decrease in the rise time of mechanical transmission through the ossicular chain.

* Discharge rates of single units in the IC do not appear to reach adult levels until some time after this30.

42 Audiological implications The results presented here, a n d in a previous study 29 o f A P a u d i o g r a m s , suggest t h a t p e r c e p t i o n o f s u p r a t h r e s h o l d , high frequency s o u n d m a y n o t develop as quickly as thresholds for s o u n d detection. G i v e n t h a t studies o f h u m a n BERs27, 45,46 generally use high intensity clicks o f mixed spectral content, it m a y be t h a t the a m p l i t u d e o f the B E R c o m p o n e n t s so r e c o r d e d is critically d e p e n d e n t on the precise spectral c o n t e n t o f the clicks used. Specifically, it w o u l d be p r e d i c t e d f r o m the present results t h a t a m p l i t u d e d a t a o b t a i n e d f r o m clicks having m o r e high-frequency c o m p o n e n t s w o u l d show r e t a r d e d d e v e l o p m e n t relative to d a t a collected using clicks o f low-frequency content. Since click-evoked B E R a m p l i t u d e is now being used as an index o f the m a t u r a t i o n a l state o f the a u d i t o r y p a t h w a y , b o t h in h u m a n s 27 a n d animals 47, it is i m p o r t a n t t h a t b o t h the intensity a n d frequency c o n t e n t o f the stimuli used in such w o r k be accurately specified a n d considered as critical variables. ACKNOWLEDGEMENTS I wish to t h a n k Dr. D. R. F. Irvine for the use o f his l a b o r a t o r y in the D e p a r t ment o f Psychology, M o n a s h University. The D e p a r t m e n t o f Psychology, University o f M e l b o u r n e p r o v i d e d me with financial s u p p o r t d u r i n g the course o f these experiments. Dr. D. P. Phillips p a r t i c i p a t e d in some o f the early experiments o f this study. Dr. G. G. R. G r e e n a n d Dr. M. H. G o l d s t e i n p r o v i d e d helpful c o m m e n t s on an earlier d r a f t o f the manuscript.

REFERENCES 1 Aitkin, L. M. and Moore, D. R., Inferior colliculus iI. Development of tuning characteristics and tonotopic organisation in central nucleus of the neonatal cat, J. Neurophysiol., 38 (1975) 1208-1216. 2 Alford, B. R. and Ruben, R. J., Physiological, behavioural and anatomical correlates of the development of hearing in the mouse, Ann. Otol. (St. Louis), 72 (1963) 237-247. 3 ~,ngg~.rd, L., An electrophysiologicalstudy of the development of cochlear functions in the rabbit, Acta oto-laryng. (Stockh.). Supplement 203 (1965) 1-64. 4 Brugge, J. F., Javel, E. and Kitzes, L. M., Signs of functional maturation of the peripheral auditory system in discharge patterns of neurons in the anteroventral cochlear nucleus of kitten, J. Neurophysiol., 41 (1978) 1557-1579. 5 Carlier, E., Abonnenc, M. and Pujol, R., Maturation des r6ponses unitaires/t la stimulation tonale dans le nerf cochl6aire du chaton, J. Physiol. (Paris), 70 (1975) 129-138. 6 Carlier, E. and Pujol, R., Role of inner and outer hair cells in coding sound intensity: an ontogenetic approach, Brain Research, 147 (1978) 174-176. 7 Crowley, D. E. and Hepp-Raymond, M. C., Development of cochlear function in the ear of the infant rat, J. comp. physiol. Psychol., 62 (1966) 427432. 8 Dallos, P., The Auditory Periphery-Biophysics and Physiology, Academic Press, New York, 1973. 9 Davis, H., Mechanisms of excitation of auditory nerve impulses. In G. L. Rasmussen and W. F. Windle (Eds.), Neural Mechanisms of the Auditory and Vestibular Systems, Charles C. Thomas, Springfield, II1., 1960. 10 Davis, H., Peripheral coding of auditory information. In W. A. Rosenblith (Ed.), Sensory Communication, Wiley, New York, 1961. 11 Finck, A., Schneck, C. D. and Hartman, A. F., Development of cochlear function in the neonate mongolian gerbil (Meriones unguiculatus), J. comp. physiol. Psychol., 78 (1972) 375-380.

43 12 Galambos, R. and Hecox, K., Clinical applications of the brain stem auditory evoked potentials. In J. Desmedt (Ed.), Progress in Clinical Neurophysiology, S. Karger, New York, 1977. 13 Gibson, M. M., Hind, J. E., Kitzes, L. M. and Rose, J. E., Estimation of travelling wave parameters from the response properties of cat AVCN neurons. In E. F. Evans and J. P. Wilson (Eds.), Psychophysics and Physiology of Hearbzg, Academic Press, London, 1977. 14 Gourevitch, G. and Hack, M. H., Audibility in the rat, J. comp. physiol. Psychol., 62 (1966) 289-291. 15 Greenwood, D. D., Critical bandwidth and the frequency co-ordinates of the basilar membrane, J. acoust. Soc. Amer., 33 (1961) 1344-1356. 16 Irvine, D. R. F., Acoustic properties of neurons in posteromedial thalamus of cat, J. Neurophysiol., 43 (1980) 395-408. 17 Jewett, D. L. and Romano, M. N., Neonatal development of auditory system potentials averaged from the scalp of rat and cat, Brain Research, 36 (1972) 101-115. 18 Kiang, N., Y.-S, Watanabe, T., Thomas, E. C. and Clarke, L. F., Discharge Patterns of Single Fibers in the Cat's Auditory Nerve, M.I.T. Press, Cambridge, Mass., 1965. 19 Liberman, M. C., Auditory-nerve response from cats raised in a low noise chamber, J. acoust. Soc. Amer., 63 (1978) 442-455. 20 Klyavina, M. P. and Maruseva, A. M., The electrical responses of the cochlea of newborn animals, Dokl. Akad. Nauk. SSSR, Otd. Biol., 149 (1963) 1221-1224. 21 Mair, I. W. S., Elverland, H. H. and Laukli, E., Development of early auditory-evoked responses in the cat, Audiology, 17 (1978) 469-488. 22 Mair, I. W. S., Elverland, H. H. and Laukli, E., Early auditory-evoked responses in the cat: rate effects, Audiology, 18 (1979) 265-278. 23 Manley, G. A., Some aspects of the evolution of hearing in vertebrates, Nature, (Lond.), 230 (1971) 506-509. 24 McCrady, E., Wever, E. G. and Bray, C. W., The development of hearing in the opossum, J. exp. ZooL, 75 (1937) 503-517. 25 McCrady, E., Wever, E. G. and Bray, C. W., A further investigation of the development of hearing in the opossum, J. comp. Psychol., 30 (1940) 17-21. 26 Mikaelian, D. and Ruben, R. J., Development of hearing in the normal 'cba-j' mouse, Acta otolaryng. (Stockh.), 59 (1965) 451-461. 27 Mokotoff, B., Schulman-Galambos, C. and Galambos, R., Brain stem auditory evoked responses in children, Arch. Otolaryng., 103 (1977) 38-43. 28 Moore, D. R., Development of the Cat Auditory System, Ph.D. Thesis, Monash University, 1978. 29 Moore, D. R. and Irvine, D. R. F., The development of some peripheral and central auditory responses in the neonatal cat, Brain Research, 163 (1979) 49-59. 30 Moore, D. R. and lrvine, D. R. F., Development of binaural input, response patterns, and discharge rate in single units of the cat inferior colliculus, Exp. Brain Res., 38 (1980) 103-108. 31 Ozdamar, O., Frequency dependence of tone-burst-generated whole nerve action potentials, J. acoust. Soc. Amer., Suppl. 1, 57 (1975) 561-562. 32 Ozdamar, O. and Dallos, P., Input-output functions of cochlear whole-nerve action potentials. interpretation in terms of one population of neurons, J. acoust. Soc. Amer., 59 (1976) 143-147. 33 Puj°l'R"Maturati°n P°smatale du Systdme Auditif Chez le Chat.l~tude Fonctionnelle et Structurale, Th6se Doct., Universit~ de Montpellier, 1971. 34 Pujol, R., Carlier, E. and Devigne, C., Different patterns of cochlea innervation during the development of the kitten, J. comp. Neurol., 177 (1978) 529-536. 35 Pujol, R. and Hilding, D., Anatomy and physiology of the onset of auditory function, Acta otolaryng. (Stockh.), 76 (1973) 1-10. 36 Pujol, R. and Marry, R., Structural and physiological relationships of the maturing auditory system. In L. Jilek and S. Trojan (Eds.), Ontogenesis of the Brabz, Charles University, Prague, 1968. 37 Pujol, R. and Marry, R., Postnatal maturation in the cochlea of the cat, J. comp. Neurol., 139 (1970) 115-126. 38 Roberto, M., A topographic quantitative analysis of the post-natal bone formation in the auditory ossicles of the dog, Acta oto-laryng. (Stockh.), 81 (1976) 16--25. 39 Romand, R., Maturation des potentiels cochldaires dans la pdriode pdrinatale chez le chat et chez le cobaye, J. PhysioL (Paris), 63 (1971) 763-782. 40 Romand, R., Development of auditory nerve activity in kittens, Brain Research, 173 (1979) 554-556.

44 41 Romand, R., Granier, M. R. and Pujol, R., Potentials cochl6aires chez le foetus de cobaye dans la p6riode pr6natale, J. Physiol. (Paris), 63 (1971) 280A-281A. 42 Romand, R., Pujol, R., Konig, N. and Marty, R., Maturation electrophysiologique de la cochl6e, C. R. Acad. Sci. (Paris), 270 (1970) 2476-2479. 43 Rose, J. E., Adri~in, H. and Santib~inez, G., Electrical signs of maturation in the auditory system of the kitten, Acta neurol, lat.-amer., 3 (1957) 133-143. 44 Ruben, R. J., Elberling, C. and Salomon, G., (Eds.,), Electrocochleography, University Park Press, Baltimore, 1976. 45 Salamy, A. and McKean, C. M., Postnatal development of human brain stem potentials during the first year of life, Electroenceph. clin. Neurophysiol., 40 (1976) 418-426. 46 Salamy, A., McKean, C. M., Pettett, G. and Mendelson, T., Auditory brainstem recovery processes from birth to adulthood, Psychophysiology, 15 (1978) 214-220. 47 Shipley, C., Buchwald, J. S., Norman, R. and Guthrie, D., Brain stem evoked response development in the kitten, Brain Research, 182 (1980) 3t3-326. 48 Starr, A., Amlie, R. N., Martin, W. H. and Sanders, S., Development of auditory function in newborn infants revealed by auditory brainstem potentials, Pediatrics, 60 (1977) 83 !-837. 49 Stevens, S. S. and Davis, H., Hearing, Wiley, New York, 1938. 50 Tasaki, I. and Davis, H., Electrical responses of individual nerve elements in cochlear nucleus to sound stimulation (guinea pig), J. Neurophysiol., 18 (1955) 151-158. 51 Tasaki, I., Davis, H. and Eldredge, D. H., Exploration of cochlear potentials with a microelectrode, J. acoust. Soc. Amer., 26 (1954) 765-773. 52 Weiss, T. F., Peake, W. T. and Sohmer, H. S., Intracochlear potential recorded with micropipets. II. Responses in the cochlear scalae to tones, J. acoust. Soc. Amer., 50 (1971) 587-601. 53 Willott, J. F. and Shnerson, A., Rapid development of tuning characteristics of inferior colliculus neurons of mouse pups, Brain Research, 148 (1978) 230-233.