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The development of some peripheral and central auditory responses in the neonatal cat
Brain Research, 163 (1979) 49-59
© Elsevier/North-Holland Biomedical Press
49
T H E D E V E L O P M E N T OF SOME P E R I P H E R A L A N D C E N T R A L A U D I T O R Y RESPONSES IN T H E N E O N A T A L CAT
D. R. MOORE and D. R. F. IRVINE Neuropsyehology Laboratory, Department of Psychology, Monash University, Clayton, Victoria 3168 (Australia)
(Accepted June 22nd, 1978)
SUMMARY The development of cochlear nerve action potential thresholds at different frequencies (AP audiograms) and inferior colliculus (IC) single unit thresholds and tuning was examined in barbiturate-anaesthetized kittens. AP thresholds decreased over the whole frequency spectrum during the first 5 weeks of life. Thresholds to highfrequency stimulation remained higher in 7-week-old animals than in adults. These results are in contrast to previous reports which have suggested that the AP response and gross cochlear anatomy are mature by the end of the second week. These differences may be due to the fact that the AP audiogram technique provides a measure of the activity of discrete regions along the cochlear partition. IC units in animals younger than 3.5 weeks had significantly elevated thresholds and broader tuning than those of the adult cat. Comparison of AP audiogram and IC unit thresholds in the adult revealed that these indices show similar frequency-dependent sensitivity. The slower maturation revealed by the AP audiogram may be due to the greater number and/or synchrony of cochlear nerve discharges needed to produce the gross AP. If this were the case, perception of suprathreshold sounds might not develop as quickly as thresholds for sound detection.
INTRODUCTION The immaturity of the neonatal cat's cochlea has been demonstrated physiologically 7,18,~1,24 and anatomically%iS, z0. The cochlear microphonic (CM) first appears in response to high-intensity clicks two days before birth, and the cochlear nerve action potential (AP) appears at birth is. Initial responses are of low amplitude (for a given sound pressure level, SPL), are easily fatigued and are only elicited by a limited stimulus frequency range15, 21. By some indices (click-evoked AP latency 21,24, endocochlear potential amplitudeT), the cochlea remains functionally immature until some
50 time between the 20th and 30th day of life. Pujol and Hilding 18, on the other hand, reported that CM and AP were mature with respect to threshold and frequency range by the 12th to 15th day after birth. Detailed evidence was not presented, however, and it was one of the aims of the present study to reinvestigate the course of development of cochlear potential thresholds. Round-window recorded AP at high stimulus intensities is known to reflect the activity of fibres from only the basal turn of the cochlea 10. Measurements of this kind therefore provide evidence on the development of only a restricted region of the cochlear partition. It has recently been established, however, that round-window recorded AP to a near-threshold tonal stimulus of a given frequency reflects the activity of fibres having that best frequencyS,12,1~. If the SPL necessary to produce a specified, low-amplitude (e.g. 20-50 #V) AP is established at a number of frequencies, the resultant curve is termed an AP audiogram. It was decided that this technique would provide a means of investigating the physiological development of different regions along the cochlear partition. The central auditory pathway has been shown to be structurally immature at birth~4,z~, 25, and a postnatal decrease of response latency has been demonstrated in single cells of central auditory nuclei6,16,17, z2. Aitkin and Moore ~ demonstrated that cells in the central nucleus of the inferior colliculus (ICC) of young kittens had higher thresholds to tonal stimuli and broader tuning curves than cells from adult cats. A relatively small number of cells was examined, however, and no precise determination of the time of maturation of these response properties was possible. In the present study, results will be reported from a larger sample of neurons, and an attempt will be made to relate these findings to those collected on development of peripheral responses.
METHODS
Subjects and surgery Forty cats in 8 age ranges were used (Table I). Adult cats were anaesthetized with sodium pentobarbital (Nembutal, 40 mg/kg, i.p.); kittens were anaesthetized with ketamine hydrochloride (Ketalar, 35 mg/kg, i.m.) and Nembutal (10 mg/kg, i.p.). Supplementary doses of anesthetic were administered whenever the animal showed a withdrawal reflex to a pinch of the paw. Rectal temperature was monitored throughout and maintained at 37 ° C (kittens) or 37.5 °C (adults). Following tracheal cannulation, a midline incision was made along the length of the dorsal surface of the skull. The skin and underlying musculature were reflected bilaterally down to the level of the external auditory meatus, and back to the lambdoid crest. The meatuses were then sectioned to enable placement of transducer-probe couplers to within 1 or 2 mm of the drum. Tissue was cleared posterior to each meatus to reveal the bulls. Five mm long screws were fixed in the skull and the head was cemented to a modified Trent-Wells stereotaxic frame.
51 TABLE I Number of cats in each age range
Age (days) Number of cats
11-15 7
16-20 21-25 26-30 7 6 4
31-35 3640 4 3
50-55 Adult 2 7
Stimulating systems
Shaped tone-bursts were produced by Beyer DT48S earphones in shielded enclosures and delivered through a metal coupler terminating in a tapered speculum inserted into the meatal stub. SPLs were monitored via 7.2 cm long × 2.5 mm internal diameter probe tubes (adults; 2.2 cm long × 1.0 mm internal diameter, kittens), mounted concentrically inside the couplers, and terminating in Bruel and Kjaer (B and K) type 4134 0.5 inch condenser microphones. The two pairs (kitten and adult) of probe tube-coupler-transducer assemblies were calibrated prior to the experiment by placing the coupler ends within 1-2 mm of a B and K type 4135 0.25 inch condenser microphone in a thick-walled polyethylene coupler. Probe correction factors were calculated at 0.1 kHz steps by comparing the output of the 0.5 inch and 0.25 inch microphones. All SPLs are expressed in dB re 0.0002 dyne/sq.cm. Procedure Peripheral responses. A small (2 mm) hole was drilled in the bulla and a stainless-
steel spring electrode was placed on the round-window. Round-window potentials were amplified by Tektronix type 122 preamplifiers and displayed on an oscilloscope. Following CM click 'threshold' (20 #V criterion) determination, the output of the round-window electrode was amplified and processed by a Data General Nova Computer, programmed for signal averaging. Sound stimuli consisted of tone bursts (50 msec duration, 1 msec rise/fall time, delivered at a rate of 5/sec) of rolling phase, such that an average of the response to 100 stimuli eliminated the CM component, leaving the AP intact. For each ear tested, 18 frequencies of stimulation were used, and for each frequency a 20 #V criterion (peak amplitude of N1) was set as an arbitrary threshold. An AP audiogram was constructed from the obtained thresholds. Latency of N1 peak from stimulus onset was also recorded. Inferior colliculus unit responses. After completion of AP audiometry, the bullae were sealed with dental acrylic and a hole was drilled in the dorsal surface of the skull to reveal the occipital cortex. The inferior colliculus (IC) was exposed by aspirating the overlying cerebrum. Stimuli consisted of tone bursts (250 msec duration, 10 msec rise/fall time, delivered at a rate of 1/sec) presented monaurally to the ear contralateral to the exposed IC. Tungsten-in-glass microelectrodes 4 (impedance at 1 kHz: 2-3 M ~ ) were advanced into the dorsal surface of the IC by means of a remotely controlled stepping motor. Acoustically responsive single cells were isolated (according to the criteria of Bishop et al. 5) and tuned z6 audiovisually. Threshold criterion was a response to 50 of the stimulus presentations. In animals of each age group an attempt was made to
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sample cells whose best frequencies spanned the adult range 11. At the conclusion of the electrode penetration an electrolytic lesion (10 #A for 10 sec) was made, the animal was decapitated and its head placed in formol-saline. Following fixation, frozen sections of the IC were cut in the sagittal plane and stained with thionine. Data from areas other than ICC were discarded. For each tuning curve, the threshold at best frequency and bandwidth (Q10a0) were determined and differences between age groups were compared by means of oneway Analyses of Variance (ANOVAs) and Newman-Keuls post-hoc comparisons 27. RESULTS
Cochlear potentials AP audiograms (Fig. 1) show markedly elevated thresholds across the entire frequency range in the youngest animals tested (11-15 days). Thresholds decline progressively, but remain elevated relative to adult levels at all frequencies, up to 31-35 days. In older animals, threshold elevation is limited to frequencies above 2 kHz, and this high-frequency elevation is still apparent in the 50-55-day group. One cat tested at 115 days showed adult-like responses, and the data for this animal are included in the adult curve. Although the shape of the audiograms in Fig. 1 does not vary markedly with age, the frequency of maximum sensitivity changes from 1 kHz (11-15 days) to 7 kHz (Adults). It is around the most sensitive frequency range in the adult cat (4-12 kHz) that the greatest threshold elevation exists in the kitten groups. This is also the range in which the mature threshold levels take longest to develop (cf., 50-55-day-old cats).
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Fig. 2. AP latency functions. Curves for animals older than 25 days closely approximated that of the adults, and are omitted for clarity of presentation. Variation in AP latency with frequency during development is shown in Fig. 2. The monotonic trend of decreasing latency with increasing frequency in adult cats provides strong confirmation that round-window recorded AP at these intensity levels reflects the activity of nerve fibres originating in discrete portions of the cochlea. Latency functions from younger animals follow the same trend, although some nonmonotonicity, probably attributable to response variability, exists at higher frequencies. Of particular interest is the finding that latency is elevated in the youngest group of animals (11-15 days), but seems to have reached adult levels by the start of the fourth week. CM click thresholds were found to change relatively little from the youngest animals tested to the adults. The mean threshold in the 11-15-day group (55 dB) was only 12 dB higher than that of the adult cats. The small number of data points prohibited any statistical evaluation of change. When these data are compared with other indices of cochlear and central development (Figs. 1-5), however, the relative maturity of CM in even the youngest kittens is striking.
Inferior colliculus unit responses A total of 186 single cells from ICC were isolated long enough for their best frequency threshold to be determined. The data yield per animal was an increasing function of age with a 3-fold increase from the youngest group to the adults. The low data yield in young animals was attributable to difficulties in maintaining correct levels of anesthesia, and to sluggishness and lack of stability of cellular responses. Mean unit threshold elevation for low- (best frequency less than 3.0 kHz) and high- (best frequency greater than or equal to 3.0 kHz) frequency cells in each age group are shown in Fig. 3. The data are plotted relative to the adult mean unit
54
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AGE GROUP (days) Fig. 3. Thresholds for kitten IC units with high ( > 3 kHz) and low ( < 3 Khz) best frequencies expressed relative to adult thresholds. The number adjacent to each point is the sample size averaged to produce that point.
thresholds (low-frequency units = 26 dB SPL; high-frequency units = 13 dB SPL). ANOVAs showed highly significant differences between age groups (low-frequency units: F6,98 = 11.50, P < 0.001 ; high-frequency units: F6,94 = 9.60, P < 0.001). For low-frequency units, Newman-Keuls comparisons (Table II(a)) revealed that each of the 3 youngest groups differed significantly from the adults. Animals in the older developmental groups (greater than 25 days) did not show significant threshold elevations from the adults. High-frequency units showed a more prolonged elevation TABLE II
Q values obtained in Newman-Keulspost-hoc comparisons of each age group with adult group (a) Unit thresholds at best.frequency Age group (days)
11-15
16-20
21-25
26-30
31-35
36-40
High-frequency Low-frequency
24.9 27.5
29.1 31.1
13.1 16.2
10.1 2.5
3.1 1.3
5.8 4.7
High frequencies Low frequencies Criterion Q
~ P = 0.05P = 0.01-
9.03 11.94
12.52 16.52
(b) Unit broadness of tuning ( Qlo) Age group (days)
Criterion Q
11-15
16-20
21-25
26-30
31-35
36-40
3.7
3.7
2.6
1.8
0.7
1.4
~ P = 0.05-2.5 P = 0.01-3.3
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of threshold, each of the 4 youngest groups differing significantly from the adults (Table II(a)). Thus, the youngest animals in which there was no significant threshold elevation were the 31-35-day group. The smaller number of neurons for which broadness of tuning data were obtained precluded separate analysis of the two frequency ranges. Broadness of tuning (Qlo) of units in the ICC of the adult cat is, however, a frequency-dependent phenomenon 2. In Fig. 4 the dashed curve (open triangles) represents the mean Q10 of all units for which these data were obtained. A second curve (continuous line, closed triangles) was plotted in which the data for each group were equalized for median frequency. This was achieved by arranging the units of each group in rank order of best frequency and eliminating every second unit from the low- or high-frequency end, until a median best frequency of 2.9-3.1 kHz was attained among the remaining units. Units from the youngest 3 groups showed significantly broader tuning than units from adult cats (F6,154 ~- 3.53, P < 0.005; see Table II(b)). Units from the older groups did not differ significantly from the adults, nor did the youngest two groups differ from one another. ICC unit thresholds and mean AP thresholds are plotted as a function of frequency in Fig. 5. Each dot represents a single ICC unit (n ---- 53). The frequency spectrum employed was divided into 6 octaves (indicated by AP standard deviation bars in Fig. 5), and within each octave the mean I C C unit threshold was calculated (open triangles, dotted lines). O f particular note is the fact that the mean unit threshold curve has the same shape as the AP threshold curve, but follows a course at about 30 dB lower intensity. These data represent further evidence of the frequency-
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Fig. 5. Comparison ofAP audiogram with unit thresholds in the adult cats. See text for explanation of symbols.
specific nature of the AP audiograms, and provide a means by which the peripheral and central acoustic responses may be compared. DISCUSSION
Cochlear potentials The AP response is generally thought to develop later than CM 3,18,21,24, but to be mature with respect to latency by about the 20th day 21. In the present study AP latency in the 16-20-day group was longer than in adults only at high frequencies, and by the start of the fourth week the latency/frequency function had attained adult levels. This finding, and that of a relatively mature CM threshold in the youngest group tested, conform with previous reports dealing with time of maturation. As was outlined in the introduction, AP threshold development has not been studied in detail previously. Anecdotal evidencOg, is suggested, however, that AP thresholds were mature by the age of 15 days. For the stimulus/response parameters used in the present study this was clearly not the case. Development of a 20 #V criterion AP response continues well into the second month of life, particularly at high frequencies. A comparison of the shapes of the audiogram curves suggests that functional cochlear development at 15 days is greatest in the apical region, and reaches maturity in this region by 36-40 days. Maturation in the basal end of the cochlea is not complete until well after 50 days. These findings depart from anatomical studies of cat cochlear development15,19,20, since gross structural development has been shown to proceed regularly from base to apex, and to be complete in all parts of the cochlea by the 15th postnatal day.
57 Inferior colliculus unit responses
The development of unit thresholds revealed in this study is in general agreement with that reported previously by Aitkin and MooreL This development has been elaborated in the present study by the separate treatment of units having low and high best frequencies and by specification of the time of attainment of adult threshold levels. Low-frequency units ranged from a mean threshold of 57 dB in the youngest groups to 26 dB in the adult group, attaining maturity by 25 days. For units having high best frequencies, thresholds ranged from 41 dB in the youngest groups to 13 dB in the adults. These units did not attain mature thresholds until about the 30th day. Tuning of units is very broad in young animals. A previous reporO dealt with too few units to evaluate accurately when maturation of this index occurred. It has now been established that units attain adult levels of tuning sharpness by the 26-30th day of life. General discussion
The present series of experiments employed animals with a minimum age of 11 days, since it has been shown1, 24 that the kitten outer and middle ear are immature (by gross structural indices) for at least the first week of life. An examination of peripheral or central thresholds in animals younger than about 11 days would therefore be difficult to interpret. In the youngest animals tested, only very intense stimuli were effective in eliciting criterion AP responses, despite the apparent maturity of the middle ear and most of the gross cochlear structures z0. In animals of all ages, the AP audiograms were some 30 dB higher than IC unit thresholds. The more rapid development of the lowfrequency regions of the cochlea revealed by the AP audiograms was reflected in the earlier maturation of IC neurons having low best frequencies. However, IC neurons of both high and low best frequency showed mature thresholds some weeks before AP thresholds achieved adult levels. These discrepancies presumably reflect the nature of gross AP, and suggest possible answers to the question of what is developing as the audiogram approaches adult levels. The fact that all IC unit thresholds are at adult levels by 30 days must mean that some auditory nerve fibres also have adult sensitivity at this age. Detection of a gross AP requires, however, that a number of nerve fibres discharge synchronously. The development of AP audiograms after 30 days, therefore, presumably reflects an increase in either the number or synchrony of elicited auditory nerve discharges. Behaviourally, this could mean that perception of suprathreshold sounds may not develop as quickly as thresholds for sound detection. The morphological basis of this development cannot be identified on the basis of the present data. CM data from this and previous studies suggest that the hair cells and receptor potential are relatively well developed by 11-15 days. The subsequent development of AP presumably reflects changes in auditory nerve terminals or in synaptic mechanisms. The development of gross cochlear structures has been well described using light microscopyZ0. However, a recent electron microscopic study of the octopus cell area of the posteroventral cochlear nucleus25 indicates that the
58 d e v e l o p m e n t o f fine cytological structure is n o t c o m p l e t e b y the time structures at the light microscopic level a p p e a r fully d e v e l o p e d 9. A n electron m i c r o s c o p i c study o f the cochlear n e r v e - h a i r cell synapse a8, o n the o t h e r hand, failed to show r e t a r d a t i o n o f synaptic d e v e l o p m e n t over gross s t r u c t u r a l d e v e l o p m e n t in the cochlea. The reconciliation o f these various a n a t o m o p h y s i o l o g i c a l relations will require m o r e d e t a i l e d E M at various levels o f the cochlea, as well as f u r t h e r evidence o f the physiological d e v e l o p m e n t o f AP. ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Phillips for helpful c o m m e n t s K e n y o n , Mr. V. K o h o u t , Mrs. R. S. G u m b y k i n d l y p r o v i d e d
Dr. L. M. Aitkin, Dr. J. R. J o h n s t o n e a n d Mr. D. P. o n a n earlier draft o f this m a n u s c r i p t ; a n d Mrs. C. J. Sack a n d Miss. P. W a r d for technical assistance. Mr. us with various surgical requisites.
REFERENCES 1 Aitkin, L. M. and Moore, D. R., Inferior colliculus, I1. Development of tuning characteristics and tonotopic organisation in central nucleus of the neonatal cat, J. NeurophysioL, 38 (1975) 1208-1216. 2 Aitkin, L. M., Webster, W. R., Veale, J. L. and Crosby, D. C., Inferior colliculus, I. Comparison of response properties of neurons in central, pericentral and external nuclei of adult cat, J. Neurophysiol., 38 (1975) 1196-1207. 3 Alford, B. R. and Ruben, R. J., Physiological, behavioral and anatomical correlates of the development of hearing in the mouse, Ann. OtoL (St. Louis), 72 (1963) 237-247. 4 Baldwin, H. A., Frenk, S. and Lettvin, J. Y., Glass coated tungsten microelectrodes, Science, 148 (1965) 1462-1464. 5 Bishop, P. O., Burke, W. and Davis, R., The identification of single units in central visual pathways, J. PhysioL (Lond.), 162 (1962) 409-431. 6 Carlier, E., Abonnenc, M. et Pujol, R., Maturation des r6ponses unitaires gtla stimulation tonale dans le nerf cochlbaire du chaton, J. Physiol. (Paris), 70 (1975) 129-138. 7 Fernandez, C. and Hinojosa, R., Postnatal development ofendocochlear potential and stria vascularis in the cat, Acta oto-laryng. (Stockh.), 78 (1974) 173-186. 8 Johnstone, J. R., Yates, G. K. and Alder, V. A., The whole nerve action potential in guinea pig. Frequency response tuning curves, in preparation. 9 Kane, E. C., Octopus cells in the cochlear nucleus of the cat: heterotypic synapses upon homeotypic neurons, Int. J. Neurosci., 5 (1973) 251-279. 10 Kiang, N. Y-S., Watanabe, T., Thomas, E. C. and Clark, L. F., Discharge Patterns of Single Fibres in the Cat's Auditory Nerve, M.I.T. Press, Cambridge, Mass., 1965. 11 Merzenich, M. M. and Reid, M. D., Representation of the cochlea within the inferior colliculus of the cat, Brain Research, 77 (1974) 397-415. 12 Ozdamar, O., Frequency dependence of tone-burst-generated whole nerve action potentials, J. Acoust. Soc. Amer., Suppl. 1, 57 (1975) 561-562. 13 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. 14 Pujol, R., D6veloppement des r6ponses ~t la stimulation sonore dans le colliculus inf6rieur chez le chat, J. Physiol. (Paris), 61 (1969) 411-421. 15 Pujol, R.,Maturation Postnatale du Systdrne Auditif Chez le Chat. l~tude Fonctionnelle et Structutale, Th6se Doct., Universit6 de Montpellier, 1971. 16 Pujol, R., Development of tone-burst responses along the cat's auditory pathway, Acta oto-laryng. (Stockh.), 74 (1972) 383-391. 17 Pujol, R. et Granier, M. R., Maturation des r6ponses auditives dans le colliculus inf6rieur chez le chat, J. Physiol. (Paris), 60 Suppl. 525 (1968).
59 18 Pujol, R. and Hilding, D., Anatomy and physiology of the onset of auditory function, Acta otolaryng. (Stockh.), 76 (1973) 1-10. 19 Pujol, R. and Marty, R., Structural and physiological relationships of the maturing auditory system. In L. Jilek and S. Trojan (Eds.), Ontogenesis of the Brain, Charles University, Prague, 1968. 20 Pujol, R. and Marry, R., Postnatal maturation in the cochlea of the cat, J. comp. NeuroL, 139 (1970) 115-126. 21 Romand, R., Maturation des potentiels cochl6aires dans le p6riode p6rinatale chez le chat et chez le cobaye, J. PhysioL (Paris), 63 (1971) 763-782. 22 Romand, R. and Marry, R., Postnatal maturation of the cochlear nuclei in the cat: a neurophysiological study, Brain Research, 83 (1975) 225-233. 23 Romand, R., Sans, A. and Marty, R., The structural maturation of the stato-acoustic nerve in the cat, J. comp. Neurol., 170 (1976) 1-16. 24 Rose, J. E., Adrian, H. and Santibanez, G., Electrical signs of maturation in the auditory system of the kitten, Acta neuroL lat.-amer., 3 (1957) 133-143. 25 Schwartz, A. M. and Kane, E. S., Development of the octopus cell area in the cat ventral cochlear nucleus, Amer. J. Anat., 148 (1977) 1-18. 26 Thurlow, W. R., Gross, N. B., Kemp, E. H. and Lowy, K., Microelectrode studies of neural activity of cat. I. Inferior colliculus, J. Neurophysiol., 14 (1951) 289-304. 27 Winer, B. J., StatisticalPrinciples in Experimental Design, McGraw-Hill, New York, 1971.
© Elsevier/North-Holland Biomedical Press
49
T H E D E V E L O P M E N T OF SOME P E R I P H E R A L A N D C E N T R A L A U D I T O R Y RESPONSES IN T H E N E O N A T A L CAT
D. R. MOORE and D. R. F. IRVINE Neuropsyehology Laboratory, Department of Psychology, Monash University, Clayton, Victoria 3168 (Australia)
(Accepted June 22nd, 1978)
SUMMARY The development of cochlear nerve action potential thresholds at different frequencies (AP audiograms) and inferior colliculus (IC) single unit thresholds and tuning was examined in barbiturate-anaesthetized kittens. AP thresholds decreased over the whole frequency spectrum during the first 5 weeks of life. Thresholds to highfrequency stimulation remained higher in 7-week-old animals than in adults. These results are in contrast to previous reports which have suggested that the AP response and gross cochlear anatomy are mature by the end of the second week. These differences may be due to the fact that the AP audiogram technique provides a measure of the activity of discrete regions along the cochlear partition. IC units in animals younger than 3.5 weeks had significantly elevated thresholds and broader tuning than those of the adult cat. Comparison of AP audiogram and IC unit thresholds in the adult revealed that these indices show similar frequency-dependent sensitivity. The slower maturation revealed by the AP audiogram may be due to the greater number and/or synchrony of cochlear nerve discharges needed to produce the gross AP. If this were the case, perception of suprathreshold sounds might not develop as quickly as thresholds for sound detection.
INTRODUCTION The immaturity of the neonatal cat's cochlea has been demonstrated physiologically 7,18,~1,24 and anatomically%iS, z0. The cochlear microphonic (CM) first appears in response to high-intensity clicks two days before birth, and the cochlear nerve action potential (AP) appears at birth is. Initial responses are of low amplitude (for a given sound pressure level, SPL), are easily fatigued and are only elicited by a limited stimulus frequency range15, 21. By some indices (click-evoked AP latency 21,24, endocochlear potential amplitudeT), the cochlea remains functionally immature until some
50 time between the 20th and 30th day of life. Pujol and Hilding 18, on the other hand, reported that CM and AP were mature with respect to threshold and frequency range by the 12th to 15th day after birth. Detailed evidence was not presented, however, and it was one of the aims of the present study to reinvestigate the course of development of cochlear potential thresholds. Round-window recorded AP at high stimulus intensities is known to reflect the activity of fibres from only the basal turn of the cochlea 10. Measurements of this kind therefore provide evidence on the development of only a restricted region of the cochlear partition. It has recently been established, however, that round-window recorded AP to a near-threshold tonal stimulus of a given frequency reflects the activity of fibres having that best frequencyS,12,1~. If the SPL necessary to produce a specified, low-amplitude (e.g. 20-50 #V) AP is established at a number of frequencies, the resultant curve is termed an AP audiogram. It was decided that this technique would provide a means of investigating the physiological development of different regions along the cochlear partition. The central auditory pathway has been shown to be structurally immature at birth~4,z~, 25, and a postnatal decrease of response latency has been demonstrated in single cells of central auditory nuclei6,16,17, z2. Aitkin and Moore ~ demonstrated that cells in the central nucleus of the inferior colliculus (ICC) of young kittens had higher thresholds to tonal stimuli and broader tuning curves than cells from adult cats. A relatively small number of cells was examined, however, and no precise determination of the time of maturation of these response properties was possible. In the present study, results will be reported from a larger sample of neurons, and an attempt will be made to relate these findings to those collected on development of peripheral responses.
METHODS
Subjects and surgery Forty cats in 8 age ranges were used (Table I). Adult cats were anaesthetized with sodium pentobarbital (Nembutal, 40 mg/kg, i.p.); kittens were anaesthetized with ketamine hydrochloride (Ketalar, 35 mg/kg, i.m.) and Nembutal (10 mg/kg, i.p.). Supplementary doses of anesthetic were administered whenever the animal showed a withdrawal reflex to a pinch of the paw. Rectal temperature was monitored throughout and maintained at 37 ° C (kittens) or 37.5 °C (adults). Following tracheal cannulation, a midline incision was made along the length of the dorsal surface of the skull. The skin and underlying musculature were reflected bilaterally down to the level of the external auditory meatus, and back to the lambdoid crest. The meatuses were then sectioned to enable placement of transducer-probe couplers to within 1 or 2 mm of the drum. Tissue was cleared posterior to each meatus to reveal the bulls. Five mm long screws were fixed in the skull and the head was cemented to a modified Trent-Wells stereotaxic frame.
51 TABLE I Number of cats in each age range
Age (days) Number of cats
11-15 7
16-20 21-25 26-30 7 6 4
31-35 3640 4 3
50-55 Adult 2 7
Stimulating systems
Shaped tone-bursts were produced by Beyer DT48S earphones in shielded enclosures and delivered through a metal coupler terminating in a tapered speculum inserted into the meatal stub. SPLs were monitored via 7.2 cm long × 2.5 mm internal diameter probe tubes (adults; 2.2 cm long × 1.0 mm internal diameter, kittens), mounted concentrically inside the couplers, and terminating in Bruel and Kjaer (B and K) type 4134 0.5 inch condenser microphones. The two pairs (kitten and adult) of probe tube-coupler-transducer assemblies were calibrated prior to the experiment by placing the coupler ends within 1-2 mm of a B and K type 4135 0.25 inch condenser microphone in a thick-walled polyethylene coupler. Probe correction factors were calculated at 0.1 kHz steps by comparing the output of the 0.5 inch and 0.25 inch microphones. All SPLs are expressed in dB re 0.0002 dyne/sq.cm. Procedure Peripheral responses. A small (2 mm) hole was drilled in the bulla and a stainless-
steel spring electrode was placed on the round-window. Round-window potentials were amplified by Tektronix type 122 preamplifiers and displayed on an oscilloscope. Following CM click 'threshold' (20 #V criterion) determination, the output of the round-window electrode was amplified and processed by a Data General Nova Computer, programmed for signal averaging. Sound stimuli consisted of tone bursts (50 msec duration, 1 msec rise/fall time, delivered at a rate of 5/sec) of rolling phase, such that an average of the response to 100 stimuli eliminated the CM component, leaving the AP intact. For each ear tested, 18 frequencies of stimulation were used, and for each frequency a 20 #V criterion (peak amplitude of N1) was set as an arbitrary threshold. An AP audiogram was constructed from the obtained thresholds. Latency of N1 peak from stimulus onset was also recorded. Inferior colliculus unit responses. After completion of AP audiometry, the bullae were sealed with dental acrylic and a hole was drilled in the dorsal surface of the skull to reveal the occipital cortex. The inferior colliculus (IC) was exposed by aspirating the overlying cerebrum. Stimuli consisted of tone bursts (250 msec duration, 10 msec rise/fall time, delivered at a rate of 1/sec) presented monaurally to the ear contralateral to the exposed IC. Tungsten-in-glass microelectrodes 4 (impedance at 1 kHz: 2-3 M ~ ) were advanced into the dorsal surface of the IC by means of a remotely controlled stepping motor. Acoustically responsive single cells were isolated (according to the criteria of Bishop et al. 5) and tuned z6 audiovisually. Threshold criterion was a response to 50 of the stimulus presentations. In animals of each age group an attempt was made to
52 110 100 _J (3_ U~
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FREQUENCY (kHz) Fig. l. AP audiograms for each age group. Each data point in this and the following figure is the mean of readings from at least 4 ears.
sample cells whose best frequencies spanned the adult range 11. At the conclusion of the electrode penetration an electrolytic lesion (10 #A for 10 sec) was made, the animal was decapitated and its head placed in formol-saline. Following fixation, frozen sections of the IC were cut in the sagittal plane and stained with thionine. Data from areas other than ICC were discarded. For each tuning curve, the threshold at best frequency and bandwidth (Q10a0) were determined and differences between age groups were compared by means of oneway Analyses of Variance (ANOVAs) and Newman-Keuls post-hoc comparisons 27. RESULTS
Cochlear potentials AP audiograms (Fig. 1) show markedly elevated thresholds across the entire frequency range in the youngest animals tested (11-15 days). Thresholds decline progressively, but remain elevated relative to adult levels at all frequencies, up to 31-35 days. In older animals, threshold elevation is limited to frequencies above 2 kHz, and this high-frequency elevation is still apparent in the 50-55-day group. One cat tested at 115 days showed adult-like responses, and the data for this animal are included in the adult curve. Although the shape of the audiograms in Fig. 1 does not vary markedly with age, the frequency of maximum sensitivity changes from 1 kHz (11-15 days) to 7 kHz (Adults). It is around the most sensitive frequency range in the adult cat (4-12 kHz) that the greatest threshold elevation exists in the kitten groups. This is also the range in which the mature threshold levels take longest to develop (cf., 50-55-day-old cats).
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Fig. 2. AP latency functions. Curves for animals older than 25 days closely approximated that of the adults, and are omitted for clarity of presentation. Variation in AP latency with frequency during development is shown in Fig. 2. The monotonic trend of decreasing latency with increasing frequency in adult cats provides strong confirmation that round-window recorded AP at these intensity levels reflects the activity of nerve fibres originating in discrete portions of the cochlea. Latency functions from younger animals follow the same trend, although some nonmonotonicity, probably attributable to response variability, exists at higher frequencies. Of particular interest is the finding that latency is elevated in the youngest group of animals (11-15 days), but seems to have reached adult levels by the start of the fourth week. CM click thresholds were found to change relatively little from the youngest animals tested to the adults. The mean threshold in the 11-15-day group (55 dB) was only 12 dB higher than that of the adult cats. The small number of data points prohibited any statistical evaluation of change. When these data are compared with other indices of cochlear and central development (Figs. 1-5), however, the relative maturity of CM in even the youngest kittens is striking.
Inferior colliculus unit responses A total of 186 single cells from ICC were isolated long enough for their best frequency threshold to be determined. The data yield per animal was an increasing function of age with a 3-fold increase from the youngest group to the adults. The low data yield in young animals was attributable to difficulties in maintaining correct levels of anesthesia, and to sluggishness and lack of stability of cellular responses. Mean unit threshold elevation for low- (best frequency less than 3.0 kHz) and high- (best frequency greater than or equal to 3.0 kHz) frequency cells in each age group are shown in Fig. 3. The data are plotted relative to the adult mean unit
54
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thresholds (low-frequency units = 26 dB SPL; high-frequency units = 13 dB SPL). ANOVAs showed highly significant differences between age groups (low-frequency units: F6,98 = 11.50, P < 0.001 ; high-frequency units: F6,94 = 9.60, P < 0.001). For low-frequency units, Newman-Keuls comparisons (Table II(a)) revealed that each of the 3 youngest groups differed significantly from the adults. Animals in the older developmental groups (greater than 25 days) did not show significant threshold elevations from the adults. High-frequency units showed a more prolonged elevation TABLE II
Q values obtained in Newman-Keulspost-hoc comparisons of each age group with adult group (a) Unit thresholds at best.frequency Age group (days)
11-15
16-20
21-25
26-30
31-35
36-40
High-frequency Low-frequency
24.9 27.5
29.1 31.1
13.1 16.2
10.1 2.5
3.1 1.3
5.8 4.7
High frequencies Low frequencies Criterion Q
~ P = 0.05P = 0.01-
9.03 11.94
12.52 16.52
(b) Unit broadness of tuning ( Qlo) Age group (days)
Criterion Q
11-15
16-20
21-25
26-30
31-35
36-40
3.7
3.7
2.6
1.8
0.7
1.4
~ P = 0.05-2.5 P = 0.01-3.3
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36-40
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AGE GROUP (days) Fig. 4. Sharpness of IC unit tuning as a function of age. The number adjacent to each point is the sample size. 'Raw' and 'Corrected' data are defined in the text.
of threshold, each of the 4 youngest groups differing significantly from the adults (Table II(a)). Thus, the youngest animals in which there was no significant threshold elevation were the 31-35-day group. The smaller number of neurons for which broadness of tuning data were obtained precluded separate analysis of the two frequency ranges. Broadness of tuning (Qlo) of units in the ICC of the adult cat is, however, a frequency-dependent phenomenon 2. In Fig. 4 the dashed curve (open triangles) represents the mean Q10 of all units for which these data were obtained. A second curve (continuous line, closed triangles) was plotted in which the data for each group were equalized for median frequency. This was achieved by arranging the units of each group in rank order of best frequency and eliminating every second unit from the low- or high-frequency end, until a median best frequency of 2.9-3.1 kHz was attained among the remaining units. Units from the youngest 3 groups showed significantly broader tuning than units from adult cats (F6,154 ~- 3.53, P < 0.005; see Table II(b)). Units from the older groups did not differ significantly from the adults, nor did the youngest two groups differ from one another. ICC unit thresholds and mean AP thresholds are plotted as a function of frequency in Fig. 5. Each dot represents a single ICC unit (n ---- 53). The frequency spectrum employed was divided into 6 octaves (indicated by AP standard deviation bars in Fig. 5), and within each octave the mean I C C unit threshold was calculated (open triangles, dotted lines). O f particular note is the fact that the mean unit threshold curve has the same shape as the AP threshold curve, but follows a course at about 30 dB lower intensity. These data represent further evidence of the frequency-
56 > Z LU A
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Fig. 5. Comparison ofAP audiogram with unit thresholds in the adult cats. See text for explanation of symbols.
specific nature of the AP audiograms, and provide a means by which the peripheral and central acoustic responses may be compared. DISCUSSION
Cochlear potentials The AP response is generally thought to develop later than CM 3,18,21,24, but to be mature with respect to latency by about the 20th day 21. In the present study AP latency in the 16-20-day group was longer than in adults only at high frequencies, and by the start of the fourth week the latency/frequency function had attained adult levels. This finding, and that of a relatively mature CM threshold in the youngest group tested, conform with previous reports dealing with time of maturation. As was outlined in the introduction, AP threshold development has not been studied in detail previously. Anecdotal evidencOg, is suggested, however, that AP thresholds were mature by the age of 15 days. For the stimulus/response parameters used in the present study this was clearly not the case. Development of a 20 #V criterion AP response continues well into the second month of life, particularly at high frequencies. A comparison of the shapes of the audiogram curves suggests that functional cochlear development at 15 days is greatest in the apical region, and reaches maturity in this region by 36-40 days. Maturation in the basal end of the cochlea is not complete until well after 50 days. These findings depart from anatomical studies of cat cochlear development15,19,20, since gross structural development has been shown to proceed regularly from base to apex, and to be complete in all parts of the cochlea by the 15th postnatal day.
57 Inferior colliculus unit responses
The development of unit thresholds revealed in this study is in general agreement with that reported previously by Aitkin and MooreL This development has been elaborated in the present study by the separate treatment of units having low and high best frequencies and by specification of the time of attainment of adult threshold levels. Low-frequency units ranged from a mean threshold of 57 dB in the youngest groups to 26 dB in the adult group, attaining maturity by 25 days. For units having high best frequencies, thresholds ranged from 41 dB in the youngest groups to 13 dB in the adults. These units did not attain mature thresholds until about the 30th day. Tuning of units is very broad in young animals. A previous reporO dealt with too few units to evaluate accurately when maturation of this index occurred. It has now been established that units attain adult levels of tuning sharpness by the 26-30th day of life. General discussion
The present series of experiments employed animals with a minimum age of 11 days, since it has been shown1, 24 that the kitten outer and middle ear are immature (by gross structural indices) for at least the first week of life. An examination of peripheral or central thresholds in animals younger than about 11 days would therefore be difficult to interpret. In the youngest animals tested, only very intense stimuli were effective in eliciting criterion AP responses, despite the apparent maturity of the middle ear and most of the gross cochlear structures z0. In animals of all ages, the AP audiograms were some 30 dB higher than IC unit thresholds. The more rapid development of the lowfrequency regions of the cochlea revealed by the AP audiograms was reflected in the earlier maturation of IC neurons having low best frequencies. However, IC neurons of both high and low best frequency showed mature thresholds some weeks before AP thresholds achieved adult levels. These discrepancies presumably reflect the nature of gross AP, and suggest possible answers to the question of what is developing as the audiogram approaches adult levels. The fact that all IC unit thresholds are at adult levels by 30 days must mean that some auditory nerve fibres also have adult sensitivity at this age. Detection of a gross AP requires, however, that a number of nerve fibres discharge synchronously. The development of AP audiograms after 30 days, therefore, presumably reflects an increase in either the number or synchrony of elicited auditory nerve discharges. Behaviourally, this could mean that perception of suprathreshold sounds may not develop as quickly as thresholds for sound detection. The morphological basis of this development cannot be identified on the basis of the present data. CM data from this and previous studies suggest that the hair cells and receptor potential are relatively well developed by 11-15 days. The subsequent development of AP presumably reflects changes in auditory nerve terminals or in synaptic mechanisms. The development of gross cochlear structures has been well described using light microscopyZ0. However, a recent electron microscopic study of the octopus cell area of the posteroventral cochlear nucleus25 indicates that the
58 d e v e l o p m e n t o f fine cytological structure is n o t c o m p l e t e b y the time structures at the light microscopic level a p p e a r fully d e v e l o p e d 9. A n electron m i c r o s c o p i c study o f the cochlear n e r v e - h a i r cell synapse a8, o n the o t h e r hand, failed to show r e t a r d a t i o n o f synaptic d e v e l o p m e n t over gross s t r u c t u r a l d e v e l o p m e n t in the cochlea. The reconciliation o f these various a n a t o m o p h y s i o l o g i c a l relations will require m o r e d e t a i l e d E M at various levels o f the cochlea, as well as f u r t h e r evidence o f the physiological d e v e l o p m e n t o f AP. ACKNOWLEDGEMENTS W e w o u l d like to t h a n k Phillips for helpful c o m m e n t s K e n y o n , Mr. V. K o h o u t , Mrs. R. S. G u m b y k i n d l y p r o v i d e d
Dr. L. M. Aitkin, Dr. J. R. J o h n s t o n e a n d Mr. D. P. o n a n earlier draft o f this m a n u s c r i p t ; a n d Mrs. C. J. Sack a n d Miss. P. W a r d for technical assistance. Mr. us with various surgical requisites.
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59 18 Pujol, R. and Hilding, D., Anatomy and physiology of the onset of auditory function, Acta otolaryng. (Stockh.), 76 (1973) 1-10. 19 Pujol, R. and Marty, R., Structural and physiological relationships of the maturing auditory system. In L. Jilek and S. Trojan (Eds.), Ontogenesis of the Brain, Charles University, Prague, 1968. 20 Pujol, R. and Marry, R., Postnatal maturation in the cochlea of the cat, J. comp. NeuroL, 139 (1970) 115-126. 21 Romand, R., Maturation des potentiels cochl6aires dans le p6riode p6rinatale chez le chat et chez le cobaye, J. PhysioL (Paris), 63 (1971) 763-782. 22 Romand, R. and Marry, R., Postnatal maturation of the cochlear nuclei in the cat: a neurophysiological study, Brain Research, 83 (1975) 225-233. 23 Romand, R., Sans, A. and Marty, R., The structural maturation of the stato-acoustic nerve in the cat, J. comp. Neurol., 170 (1976) 1-16. 24 Rose, J. E., Adrian, H. and Santibanez, G., Electrical signs of maturation in the auditory system of the kitten, Acta neuroL lat.-amer., 3 (1957) 133-143. 25 Schwartz, A. M. and Kane, E. S., Development of the octopus cell area in the cat ventral cochlear nucleus, Amer. J. Anat., 148 (1977) 1-18. 26 Thurlow, W. R., Gross, N. B., Kemp, E. H. and Lowy, K., Microelectrode studies of neural activity of cat. I. Inferior colliculus, J. Neurophysiol., 14 (1951) 289-304. 27 Winer, B. J., StatisticalPrinciples in Experimental Design, McGraw-Hill, New York, 1971.