Inferior
Colliculus.
Characteristics Central De@rtmPnt
and Tonotopic
Nucleus
L. M. AITKIN
AND
of Physiology,
II. Development
of Tuning
Organization
of the Neonatal
in
Cat
D. R. MOORE Monash
University,
Clayton,
Victoria
3 168,
,4ustralia
NEURONS IN the inferior colliculus of the frequency thresholds of single units imadult cat show different response charac- prove in the weeks following birth. Our teristics to tonal stimuli depending on results suggest that although auditory their location in the central (ICC), peri- function may be present at birth, the recentral (ICP) or external (ICX) nuclei ( 1). sponses of inferior colliculus units take between 3 and 4 weeks to acquire adult Neurons in ICC are strictly tonotopically characteristics. organized (14) and display characteristic V-shaped tuning curves whose sharpness increases with best frequency (1). Units in METHODS ICP and ICX are more broadly tuned and Considerable difficulty was experienced with appear to have other characteristics which anesthesia in this study and none of the three separate them functionally from elements anesthetic agents used ( 1% chloralose- 10% in ICC. It was of interest to examine the urethan, sodium pentobarbital, ketamine hydevelopment of neural response properdrochloride) was found to be completely reliable. Consequently, of the 20 kittens used, only ties in ICC of the postnatal cat. The development of unit responses to 12 ranging in age from 2 to 28 days, were under anesthetic for a sufficient sound stimulation in neonatal cats has maintained been the subject of a number of recent time to provide useful data relating to auditory investigations (9, 10, 13, 16, 17, 20). Al- evoked activity. In these 12 cats, seven complete penetrations were made through the inferior though the cochlea and auditory pathway colliculus in kittens of ages 6, 7, 11, 14, 17, 21, are capable of transmitting acoustic in- and 28 days. These animals were anesthetized formation at birth (10, 12, IS), the re- with an intraperitoneal injection of sodium pensponsesof single units in different regions tobarbital (10 mg/kg) and 4 of them additionally of the auditory pathway show very high received a prior intramuscular injection of (20 mg/kg). thresholds and respond only to low and ketamine hydrochloride Following anesthetic induction, the right mid-range frequencies for some weeks after birth (13, 16, 17). Discharge patterns pinna was removed and the eardrum exposed. and latencies of evoked responses to tones A tracheal cannula was inserted and the skull was cemented to a head holder attached to a also show marked changes in this period Narishige stereotaxic frame. A large section of (16, 17). the skull overlying the occipital cortex was reThree main questions relating to the moved. Cortical tissue was then aspirated by development of auditory responses were suction to expose the left superior and inferior the objects of the present study. We colliculi. The entire preparation was placed in wished to know, first, whether tonotopic a sound-attenuated room and an electric blanorganization is present in the inferior col- ket was placed around the kitten at 38°C. Unit recordings were made with glassliculus of neonatal cats; second, what connected form tuning curves take in these animals insulated tungsten microelectrodes preamplifier. After insertand how tuning curves develop with age; to a high-impedance and finally, in what manner best- ing the electrode, the opening was sealed with
Received 1208
for
publication
December
16, 1974.
3% agar gel. Tones were generated by a Bruel and Kjaer heterodyne analyzer (type 2010),
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DEVELOPMENT
OF
INFERIOR
shaped into tone pips with a locally designed and transduced by a tone-burst generator, Beyer DT48S earphone housed in a metal dome and connected to a 6-cm metal tube by a IO-cm polyethylene tube. The metal tube was placed with its opening approximately 1 mm from the eardrum. The output of a calibrated concentrically located metal probe tube and microphone (Bruel and Kjaer type 4133) was monitored by the heterodyne analyzer, thus on-line measurements of making available sound pressure level. This sound system was capable of supplying to the eardrum tones whose maximum amplitudes were in excess of 100 dB at frequencies up to 25 kHz. All auditory responses in this study were evoked by contralateral stimulation. Electrolytic lesions were made in the tissue at the termination of each successful electrode track, and in five animals a second lesion was placed at the beginning of the tonotopic sewas calculated from these quence. Shrinkage two lesions, and varied from 0 to 12% (average 5%). The animal was killed with an overdose of anesthetic and its brain was removed and placed in 10% formal-saline. Following fixation of the brain, frozen sections were cut at 50 pm in the sagittal plane and stained with cresyl violet or Thionine.
RESULTS
The inferior colliculus of the kitten can be divided into the three nuclear groups seen in the adult at least as early as 2 days. At this age, cell bodies observed in Thionine-stained sections in both ICC and ICP were small (5-10 pm) and intensely stained, with little cytoplasm. The pericentral nucleus appeared as an uneven and densely packed layer of cells with clearly defined dendrites, especially near the dorsal surface of inferior colliculus, and even under a low power of magnification was clearly separable from the central nucleus. The external nucleus contained large pale cell profiles (20-25 p) associated with numerous small darkly stained cells. Some external and middle ear structures showed marked physical changes during the first week after birth. Postmortem examination of kittens less than 6 days of age revealed the presence of viscousjellylike fluid encasing the ossiclesand occupying much of the middle ear cavity. This disappeared after the first week, but
COLLICULUS
1209
for 3-4 further days the external auditory meatus (which was always removed prior to stim ulation) was collapsed and effectively closed. In all experiments, microelectrodes were orientated to pass from the dorsal surface of the inferior colliculus to its ventralmost aspect, terminating at a level more posterior than the point of entry. This procedure allowed the full dorsoventral extent of the inferior colliculus to be sampled in each successful experiment. Of the 79 units studied, 65 units from 7 kittens, ranging in age from 6 to 28 days, provided tuning curves. As for the adult cat ( i), only a small proportion (8 units) were from the pericentral nucleus. The remainder were isolated in the central nucleus, no penetrations being made through the external nucleus. Although the number of units isolated in ICP was very small, the tuning characteristics of units in ICC and ICP appeared to be different in kittens as they have been shown to be in the adult (1). In Fig. lA, four units in ICP from cat 74-32 (21 days) and cat 74-34 (28 days) are seen to have very broad irregular tuning curves, that for 74-32-02 being particularly flat and broad, spanning at least 8 octaves. By contrast, tuning curves from the central nucleus of a younger kitten (Fig. lB, 74-31; 17 days), although clearly different to those of the adult, have a V shape and show a relatively restricted range of effective frequencies. I
Tuning
curves
In view of the smal1 number of observations for neurons in divisions outside the central nucleus, detailed attention was paid to the responses of cells in this division. In thi s studv the youngest age at whit h units showed preferential tu ning to sounds of different frequencies was 6 days. Eight units were isolated in ICC in animals 2 and 3 days of age but, although these units clearly respo nded to sound, their discha.rges tended to habit uate to stimulation. Their spike repetitive amplitudes were small and they proved difficult to isolate from background activity. Our impressions were that these discharges responded to tones without show-
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L. M. AITKIN
1210
I
I
1
AND
IIII~
D. R. MOORE
1
0.5
1.0
1
I
I~IIII~
2.0
I
5.0
10.0
FREQUENCY
1
20.0
30.0
(kHz)
07
1 1
I
1
I
I
I
I
I
I
I
I
FREQUENCY 1. Tuning curves for units in the pericentral ent symbols are used for clarity of presentation only. FIG.
ing any frequency specificity, and they had very high thresholds. Tuning curves are illustrated for units of ICC from 6-, ll- (Fig. 2A), and 14day-old kittens (Fig. 2B). The two tuning curves from the 6-day cat (74.61-1,2) demonstrate very high thresholds but are less broad than those from the 1 l-day (667,9,12; Fig. 2A) or the 14-day kitten (2% 5,7,8; Fig. 2B). In cats younger than approximately 21 days, units were only occasionally observed to fire at frequencies in excess of 10,000 Hz, even in the ventral part of the central nucleus where highfrequency responses are always observed in the adult (1, 14) (e.g., 29-10, Fig. 2B), and
nucleus,
ICP
I
III
1
1
(kHz)
(A), and central
nucleus,
ICC
(B). Differ-
their tuning curves often featured a very sudden cutoff in response at the upperfrequency limit (66-9, Fig. 2A ; 29-5, Fig. W As the postnatal age increased, tuning curves became sharper, high-frequency stimuli became more effective, and thresholds diminished (Fig. 1B). Q factors (1) were measured for 31 units of ICC in four kittens aged between 14 and 28 days. The remaining 34 tuning curves were either too irregular for Q measurements or the units were found to be located in ICP. The (2 factors were pooled and plotted with similar measurements for 92 units in ICC of the adult cat, described in the previous
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DEVELOPMENT
OF
INFERIOR
COLLICULUS
A
1211
l-2
X
80
70
I
n
1
I
1
1
,
,,,,I
I
1
I
74-29-08
74-29-06
”
1 0’1
FIG.
(74-29)
’
1
1111 ok
’
1
1
210 FREQUENCY
2. Tuning curves for units in 6-day (74-61) and (B). Note that the ordinate for graph A is expanded
paper (Fig. 8 of ref 1) and illustrated in the histogram of Fig. 3. Inspection of this histogram reveals that nearly 70% of units in the inferior colliculus of the kitten had Q factors below 3, compared with less than 30% in
T
I
Go '
7.0
1 I
Id0
I
20'0
'3dO
(kHz)
1 l-day (74-66) kittens (A) and in a 14-day kitten compared with that for graph B. Central nucleus.
the adult. This difference is likely to be composed of two factors. First, as individual tuning curves suggested, the majority of tuning curves were flatter in the neonatal cats than in the adults. Second, units sensitive to high-frequency stimuli
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L. M.
1212
q q
[0,3)
[3,6 )
AITKIN
AND
tween different age groups, a clear tendency for thresholds to decrease from 6 days to reach low levels at 2 1 and 28 days was associated with little dependence on best frequency between 500 and 20,000 Hz. These data were pooled for each age group, converted from decibel values to dynes per square centimeter, means and standard deviations were determined and reconverted to decibel values, and the results were plotted in Fig. 5. Thresholds at 6 and 7 days of age were based on measurements of only three tuning curves but have been included since they are similar in magnitude to the means for the 12 tuning curves at day Il. After this date, a precipitous drop in mean threshold occurred which stabilized near the adult value at day 21. Adult values were computed from material included in the preceding paper (1).
ADULT KITTEN
1
[6,9)
[9,12)
VALUE
OF
[l&15)
'15
0
FIG. 3. Histogram of Q values for tuning curves from the central nucleus of the inferior colliculus in adult cats and kittens. N = 3 1 for kittens, N = 92 for adults.
were less common the adult.
in the kitten
than
D. R. MOORE
in
Thresholds
Tonotopic
The most marked change in the measured parameters as a function of age was in the thresholds of units at their best frequencies. These are plotted for six kittens as a function of best frequency in Fig. 4. Although there is considerable overlap be-
Penetrations through the inferior colliculus of kittens began with the recording of units and background activity which were broadly and irregularly tuned. This pattern persisted throughout the penetration with 6- and 7-day kittens, but was re-
$20-u
organization
-
ZlOO-
z w
0
-
0 m
2 w 8~ f
m
0
l 0
-
Icn W
60-
I-
0;
m
l
0
‘3
0 o
0
0 0
_
I
0.1
I
I~IIII~
r
0.5
1.0
1
2.0 BEST
I
7
A
1’1111
5.0 FREQUENCY
FIG. 4. Scatter plot of best-frequency thresholds of units in six kittens days, w = 11 days, l = 14 days, o = 17 days, A = 21 days, A = 28 days.
.
as a function
0
1
100 (kHz)
I
2oa
of frequency.
300 q
= 6
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DEVELOPMENT
0
FIG.
standard groups
4
5.
8
12
16
AGE
(DAYS)
20
Mean threshold deviations, plotted of cats. -
OF
24
28
INFERIOR
COLLICULUS
1213
placed by the occurrence of units with best frequencies which became progressively higher as the electrode penetrated further, in kittens older than 11 days. The sequences of units with increasing best frequencies obtained in penetrations from ll-, 14-, 17-, and 21-day kittens are plotted in Fig. 6 as a function of relative location of units within a given penetration in 0.5-mm steps. The start of each sequence has been arbitrarily located on the ordinate only for clarity of presentation. Each penetration traversed the entire dorsoventral extent of the central nucleus. A number of features of tonotopic organization are apparent in this illustration. First, for lower frequencies the trends exhibited for each cat were similar and the 3 octaves from 500 to 4,000 Hz were represented in the first 1.2-1.5 mm of the central nucleus. Second, both the number of observations with best frequencies in the 3 octaves above 4,000 Hz and the upper frequency limit increased
ADULT
in decibels SPL, with against age for eight - age-
A
A A
A
0
0
11 DAY
0 0
0 0
0
0
14 DAY 0
0 0
8 o
0
17
0
DAY
0
X It X
21
DAY X x
I
0.3
’
I”“1
0.5
I
1.0
I
2.0 3.0 FREQUENCY
’
I”“1
5-o (kHz)
10.0
x
1
1
2o”o
30.0
FIG. 6. Best frequency in kilohertz (abscissa) as a function of depth from the penetration point at which a tonotopic sequence was first encountered, in 0.5-mm steps (ordinate). The sequence for each cat has been commenced at a different point on the ordinate for clarity of presentation only. Each penetration traversed the entire dorsoventral extent of the central nucleus.
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L. M.
1214
AITKIN
AND
with age. Although it was not quantified, we also noted that the anatomical size of the inferior colliculus increased in this period. Third, with the 17- and 21-day kittens, a number of units were encountered with best frequencies deviating from the trend for the majority of observations. The latter finding could be explained if or fibers were - occasionstellate neurons ally encountered, but it could also be indicdevelop ment of the ative of continual organization of the central nucleus. DISCUSSION
The results presented suggest that the inferior colliculus in the neonatal cat, alto auditory stimuli though responsive soon after birth (10, 16), undergoes substantial maturation in the subsequent 34 wk of life. It would appear that -neuronal thresholds improve from a mean of near 100 dB SPL to a value of about 40 dB SPL 2 wk and over a period of approximately to highthat, concom itan tly , responses more frequency tonal stimuli become common’ although, in view of the small sample size, some caution is needed in vieiing this latter observation. It would bealso seem that an orderly relationship tween the best frequency of a neuron and its location in the centr #al nucleus exists as soon as neurons show selectivitv in their response to the frequency of tonal stimuli. to those of These results are similar from neurons Konishi (11) who recorded in the cochlear nuclei of duck embryos. His study indicated that high bestfrequency neurons appeared after ‘units responding to low frequencies were recorded, that thresholds improved with increasing developmental age, and that tonotopic organization was present at an early age. Relationship
to cochlear
maturation
Some of these results in the cat have been hinted at in previous studies, particularly in those of Pujol (16, 17) and of Maruseva and Malysheva (13). The limited frequency-response ranges of units in very young cats have been found by Pujol to extend from 250 to 2,500 Hz in 2- to S-day-old kittens to more adult values in cats beyond the first week of age (16, 17). It is not clear whether the increase in fre-
D. R. MOORE
quency range is due to cochlear development or to neural maturation, since there has been no equivalent study performed on the development of tuning in cochlear nerve fibers. However, the cochlea shows anatomically adult development 10 days after birth in the cat (18) and cochlear microphonic potentials may be elicited over a wide frequency range in the 6-day rabbit (2). Although such information might suggest that the development of tuning and the diminution of thresholds of inferior colliculus neurons succeed the full development of the cochlea, the recent study of Fernandez and Hinojosa (6) has indicated that the stria vascularis reaches maturity at about the 25th postnatal day and parallels the adult development of the endocochlear potential by the 27-29th day. These times are of a similar order to those for the leveling of response thresholds to adult values in the inferior colliculus. There is little doubt that the neural auditory system of the cat is capable of transmitting some information at birth (12), and unit responses have been detected in the auditory cortex 24 h thereafter (10). However, the elaboration of a tonotopic framework in the inferior colliculus, clear at 11 days, appears to lag behind the structural development of the organ of Corti. It is interesting that many authors (3, 19) have shown that, morphologically, cochlear development proceeds from base to apex. This would appear to contradict our observations that initial evoked activity in inferior colliculus is tuned to a frequency range which would correspond to the apical and middle regions of the organ of Corti. Furthermore, behavioral responses are first initiated to low-frequency tones and are induced by higher tones as much as 4 days after low-tone responses (7). A similar picture exists for the development of brain stem-evoked reponses in the chicken, which show a systematic increase in sensitivity toward higher frequencies as a function of age (23), as do single units in the duck embryo (11). Our results are similar to these, and indicate that unit restimulation sponses to high-frequency (20-30 kHz) become more common in kittens 34 wk in age and, at the same time, the inferior colliculus increases in size.
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DEVELOPMENT
OF
INFERIOR
This discrepancy between cochlear morphology and auditory physiology might be explained if the functional development of the cochlea proceeded from the transmission of low frequencies to the signaling of higher frequencies at a later age. It is probable that the fluid present in the kitten middle ear for the first week of life would impede the reception of higher frequency sounds by the cochlea. The disappearance of such a middle ear impedance could facilitate the development of the high-frequency responsiveness of the cat auditory system. Neural maturation It is possible that cochlear development may also be in part responsible for the decrease in best-frequency thresholds during the first 2-3 wk of life. However, two neural processes, which have been shown to change after birth, may contribute largely to these threshold alterations. First, axons in the inferior colliculus of neonatal cats are poorly myelinated at birth, and myelination proceeds to the adult stage over the subsequent 34 wk of life (16). Such axons would be expected to exhibit low conduction velocities compared with the adult, leading to longer latencies of response in the inferior colliculus. Latencies of evoked potentials have been shown to decrease with age at several levels of the auditory pathway (16, 17, 21). It is also well known that the timing of unit discharges in the adult inferior colliculus is very precise (8, 22). It is likely that such precise timing would be blurred of slowly conducting in a population axons if it required the convergence of information, contained in impulses carried by several such axons, on single inferior colliculus neurons. A further consequence of asynchronous input to a given cell from a population of slowly conducting axons is likely to be an elevation of the thresholds of the responses of inferior colliculus cells. A second consideration relating to threshold changes is that synaptic density is likely to increase after birth in the inferior colliculus as it does in the cat visual cortex (5) where the main phase of synaptic development is critically dependent on
COLLICULUS
1215
function. It is possible that decreases in response threshold seen after the clearing of the middle ear are due to a stimulation of synaptic development in neural auditory structures by the increasing exposure of the auditory pathway to sounds of significance to the developing cat. That such continual development occurs is indicated behaviorally by the observations that, although the inception of hearing occurs in kittens at 5 days (7), more complex responses to sound such as auditory orientating and auditory following responses may take as many as 16 days longer to develop (15). These acousticomotor reflexes related to sound localization are likely to depend on the adult development of the inferior colliculus (4, 22). SUMMARY
Tuning curves were measured for 65 units in the inferior colliculus of seven anesthetized kittens aged from 6 to 28 days. At 2 days of age the inferior colliculus was divisible into central, pericentral, and external nuclei. Evidence was found for broader tuning curves to occur in the pericentral nucleus compared with the central nucleus, as has been observed in the adult. The middle ear was filled with serous fluid to 6 days, while the external auditory meatus remained collapsed until 10 days. Central nucleus tuning curves in kittens were relatively flat with high thresholds. Best-frequency thresholds diminished from a mean of near 100 dB SPL at 6-l 1 days to near 40 dB in the adult. The marked drop in thresholds between days 11 and 21 led to the adoption of the sharp form of tuning curve common for adults. of the central organization Tonotopic nucleus was clear at day 11. Speculations were advanced about the dependence of central auditory maturations on cochlear development, axon myelination in the auditory pathway, and changes in synaptic density as a function of age. ACKNOWLEDGMENTS
We particularly thank Debbi Spain and Moyra Farrington for secretarial and histological assistance. This study was supported by grants from the Australian Research Grants Committee.
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L. M.
AITKIN
AND
D. R. MOORE
REFERENCES 1. 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. Neurt$hy.siol. 38: 1196-1207, 1975. L. An electrophysiological study of 2. ANGGARD, the development of cochlear function in the rabbit. Acta Oto-Laryngol. Suppl. 203: l-64, 1965. B. J. The Temporal Bone 3. BAST, T. AND ANSON, and the Ear. Springfield, Ill.: Thomas, 1949. P., St. LAURENT, J., AND MENINI, C. H. 4. BUSER, Intervention du colliculus inferieur dans I’elaboration et le controle cortical specifique des decharges clonique au son chez le chat sur chloralose. Exptl. Brain Res. 1: 102-126, 1966. 5. CRAGG, B. G. The development of synapses in cat visual cortex. Invest. Ophthalmol. 11: 377-385, 1972. 6. FERNANDEZ, C. AND HIN~JOSA, R. Postnatal development of endocochlear potential and stria vascularis in the cat. Acta Oto-Laryngol. 78: 173-186, 1974. 7. Foss, I. AND FLOTTORP, G. A comparative study of the development of hearing and vision in various species commonly used in experiments. Acta Oto-Laryngol. 77: 202-214, 1974. 8. HIND, J. E., GOLDBERG, J. M., GREENWOOD, D. D., AND Rose, J. E. Some discharge characteristics of single neurons in the inferior colliculus of the cat. II. Timing of the discharges and observations on binaural stimulation. J. Neurophysiol. 26: 32 l-34 1, 1963. 9. KONIC;, N. AND MARTY, R. On functions and structure of deep layers of immature auditory cortex. J. Physiol., Paris 68: 145-155, 1974. 10. K~NIG, N., PUJOL, R., AND MAR*TY, R. A laminar study of evoked potentials and unit responses in the auditory cortex of the postnatal cat. Brain Res. 36: 469-473, 1972. 11. KONISHI, M. Development of auditory neuronal responses in avian embryos. Proc. Natl. Acad. Sci. U.S. 70: 17954798, 1973. 12. MARTY, R. AND T~IOMAS, J. Reponse electrocorticale a la stimulation du nerf cochleaire chez le
chat nouveau-n&J. Physiol., Paris 55: 165-166, 1963. 13. MARUSEVA, A. M. AND MALYSHEVA, V. I. Effects of acoustic stimuli on unit activity of inferior colliculus in cats. Zh. Evol. Biokhim. Fiziol. 6: 225-234, 1970. Translated in: Neurosci. Transl. 15: 41-49, 1970-1971. 14. MERZENICH, M. M. AND REID, M. D. Representation of the cochlea within the inferior colliculus of the cat. Brain Res. 77: 397-415, 1974. 15. NORTON, T. T. ReEeptive-field properties of superior colliculus cells and development of VISual behavior in kittens. J. Neurqbhysiol. 37: 674690, 1974. 16. PUJOL, R. Maturation postnatale du systeme auditif chex le chat. Etude fonctionelle et structurale (These Doct.). Montpellier: Etat. Sci. Nat., 197 1. of tone burst responses 17. PUJOL, R. Development along the auditory pathway in the cat. Acta Oto-Laryngol. 74: 383-39 1, 1972. 18. PUJOL, R. AND HILDIN~;, D. Anatomy and physiology of the onset of auditory function. Acta Oto-Laryngol. 76: l-10, 1973. 19. PUJOL, R. AND MARTY, R. Postnatal maturation of the cochlea of the cat. J. Camp. Neurol. 139: 115-126, 1970. 20. ROMAND, R., GRANIER, M. R., AND MARTY, R. Developpment postnatal de l’activite provoquee dans I’olive superieure laterale chez le chat par la stimulation sonore. J. Physiol., Paris 66: 303315, 1973. H., AND SANTIB~NEZ, G. 21, ROSE, J. E., ADRIAN, Electrical signs of maturation in the auditory system of the kitten. Acta. Neurol. Latinoam. 3: 133-143, 1957. C. D., AND 22. ROSE, J. E., GROSS, N. B., GEISLER, HIND, J. E. Some neural mechanisms in the inferior colliculus of the cat which may be relevant to localization of a sound source. J. Neurophysiol. 29: 288-3 14, 1966. J. C., COLES, R. B., AND GATES, G. R. 23. SAUNDERS, The development of auditory evoked responses in the cochlea and cochlear nuclei of the chick. Brain Res. 63: 59-74, 1973.
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Colliculus.
Characteristics Central De@rtmPnt
and Tonotopic
Nucleus
L. M. AITKIN
AND
of Physiology,
II. Development
of Tuning
Organization
of the Neonatal
in
Cat
D. R. MOORE Monash
University,
Clayton,
Victoria
3 168,
,4ustralia
NEURONS IN the inferior colliculus of the frequency thresholds of single units imadult cat show different response charac- prove in the weeks following birth. Our teristics to tonal stimuli depending on results suggest that although auditory their location in the central (ICC), peri- function may be present at birth, the recentral (ICP) or external (ICX) nuclei ( 1). sponses of inferior colliculus units take between 3 and 4 weeks to acquire adult Neurons in ICC are strictly tonotopically characteristics. organized (14) and display characteristic V-shaped tuning curves whose sharpness increases with best frequency (1). Units in METHODS ICP and ICX are more broadly tuned and Considerable difficulty was experienced with appear to have other characteristics which anesthesia in this study and none of the three separate them functionally from elements anesthetic agents used ( 1% chloralose- 10% in ICC. It was of interest to examine the urethan, sodium pentobarbital, ketamine hydevelopment of neural response properdrochloride) was found to be completely reliable. Consequently, of the 20 kittens used, only ties in ICC of the postnatal cat. The development of unit responses to 12 ranging in age from 2 to 28 days, were under anesthetic for a sufficient sound stimulation in neonatal cats has maintained been the subject of a number of recent time to provide useful data relating to auditory investigations (9, 10, 13, 16, 17, 20). Al- evoked activity. In these 12 cats, seven complete penetrations were made through the inferior though the cochlea and auditory pathway colliculus in kittens of ages 6, 7, 11, 14, 17, 21, are capable of transmitting acoustic in- and 28 days. These animals were anesthetized formation at birth (10, 12, IS), the re- with an intraperitoneal injection of sodium pensponsesof single units in different regions tobarbital (10 mg/kg) and 4 of them additionally of the auditory pathway show very high received a prior intramuscular injection of (20 mg/kg). thresholds and respond only to low and ketamine hydrochloride Following anesthetic induction, the right mid-range frequencies for some weeks after birth (13, 16, 17). Discharge patterns pinna was removed and the eardrum exposed. and latencies of evoked responses to tones A tracheal cannula was inserted and the skull was cemented to a head holder attached to a also show marked changes in this period Narishige stereotaxic frame. A large section of (16, 17). the skull overlying the occipital cortex was reThree main questions relating to the moved. Cortical tissue was then aspirated by development of auditory responses were suction to expose the left superior and inferior the objects of the present study. We colliculi. The entire preparation was placed in wished to know, first, whether tonotopic a sound-attenuated room and an electric blanorganization is present in the inferior col- ket was placed around the kitten at 38°C. Unit recordings were made with glassliculus of neonatal cats; second, what connected form tuning curves take in these animals insulated tungsten microelectrodes preamplifier. After insertand how tuning curves develop with age; to a high-impedance and finally, in what manner best- ing the electrode, the opening was sealed with
Received 1208
for
publication
December
16, 1974.
3% agar gel. Tones were generated by a Bruel and Kjaer heterodyne analyzer (type 2010),
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DEVELOPMENT
OF
INFERIOR
shaped into tone pips with a locally designed and transduced by a tone-burst generator, Beyer DT48S earphone housed in a metal dome and connected to a 6-cm metal tube by a IO-cm polyethylene tube. The metal tube was placed with its opening approximately 1 mm from the eardrum. The output of a calibrated concentrically located metal probe tube and microphone (Bruel and Kjaer type 4133) was monitored by the heterodyne analyzer, thus on-line measurements of making available sound pressure level. This sound system was capable of supplying to the eardrum tones whose maximum amplitudes were in excess of 100 dB at frequencies up to 25 kHz. All auditory responses in this study were evoked by contralateral stimulation. Electrolytic lesions were made in the tissue at the termination of each successful electrode track, and in five animals a second lesion was placed at the beginning of the tonotopic sewas calculated from these quence. Shrinkage two lesions, and varied from 0 to 12% (average 5%). The animal was killed with an overdose of anesthetic and its brain was removed and placed in 10% formal-saline. Following fixation of the brain, frozen sections were cut at 50 pm in the sagittal plane and stained with cresyl violet or Thionine.
RESULTS
The inferior colliculus of the kitten can be divided into the three nuclear groups seen in the adult at least as early as 2 days. At this age, cell bodies observed in Thionine-stained sections in both ICC and ICP were small (5-10 pm) and intensely stained, with little cytoplasm. The pericentral nucleus appeared as an uneven and densely packed layer of cells with clearly defined dendrites, especially near the dorsal surface of inferior colliculus, and even under a low power of magnification was clearly separable from the central nucleus. The external nucleus contained large pale cell profiles (20-25 p) associated with numerous small darkly stained cells. Some external and middle ear structures showed marked physical changes during the first week after birth. Postmortem examination of kittens less than 6 days of age revealed the presence of viscousjellylike fluid encasing the ossiclesand occupying much of the middle ear cavity. This disappeared after the first week, but
COLLICULUS
1209
for 3-4 further days the external auditory meatus (which was always removed prior to stim ulation) was collapsed and effectively closed. In all experiments, microelectrodes were orientated to pass from the dorsal surface of the inferior colliculus to its ventralmost aspect, terminating at a level more posterior than the point of entry. This procedure allowed the full dorsoventral extent of the inferior colliculus to be sampled in each successful experiment. Of the 79 units studied, 65 units from 7 kittens, ranging in age from 6 to 28 days, provided tuning curves. As for the adult cat ( i), only a small proportion (8 units) were from the pericentral nucleus. The remainder were isolated in the central nucleus, no penetrations being made through the external nucleus. Although the number of units isolated in ICP was very small, the tuning characteristics of units in ICC and ICP appeared to be different in kittens as they have been shown to be in the adult (1). In Fig. lA, four units in ICP from cat 74-32 (21 days) and cat 74-34 (28 days) are seen to have very broad irregular tuning curves, that for 74-32-02 being particularly flat and broad, spanning at least 8 octaves. By contrast, tuning curves from the central nucleus of a younger kitten (Fig. lB, 74-31; 17 days), although clearly different to those of the adult, have a V shape and show a relatively restricted range of effective frequencies. I
Tuning
curves
In view of the smal1 number of observations for neurons in divisions outside the central nucleus, detailed attention was paid to the responses of cells in this division. In thi s studv the youngest age at whit h units showed preferential tu ning to sounds of different frequencies was 6 days. Eight units were isolated in ICC in animals 2 and 3 days of age but, although these units clearly respo nded to sound, their discha.rges tended to habit uate to stimulation. Their spike repetitive amplitudes were small and they proved difficult to isolate from background activity. Our impressions were that these discharges responded to tones without show-
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L. M. AITKIN
1210
I
I
1
AND
IIII~
D. R. MOORE
1
0.5
1.0
1
I
I~IIII~
2.0
I
5.0
10.0
FREQUENCY
1
20.0
30.0
(kHz)
07
1 1
I
1
I
I
I
I
I
I
I
I
FREQUENCY 1. Tuning curves for units in the pericentral ent symbols are used for clarity of presentation only. FIG.
ing any frequency specificity, and they had very high thresholds. Tuning curves are illustrated for units of ICC from 6-, ll- (Fig. 2A), and 14day-old kittens (Fig. 2B). The two tuning curves from the 6-day cat (74.61-1,2) demonstrate very high thresholds but are less broad than those from the 1 l-day (667,9,12; Fig. 2A) or the 14-day kitten (2% 5,7,8; Fig. 2B). In cats younger than approximately 21 days, units were only occasionally observed to fire at frequencies in excess of 10,000 Hz, even in the ventral part of the central nucleus where highfrequency responses are always observed in the adult (1, 14) (e.g., 29-10, Fig. 2B), and
nucleus,
ICP
I
III
1
1
(kHz)
(A), and central
nucleus,
ICC
(B). Differ-
their tuning curves often featured a very sudden cutoff in response at the upperfrequency limit (66-9, Fig. 2A ; 29-5, Fig. W As the postnatal age increased, tuning curves became sharper, high-frequency stimuli became more effective, and thresholds diminished (Fig. 1B). Q factors (1) were measured for 31 units of ICC in four kittens aged between 14 and 28 days. The remaining 34 tuning curves were either too irregular for Q measurements or the units were found to be located in ICP. The (2 factors were pooled and plotted with similar measurements for 92 units in ICC of the adult cat, described in the previous
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DEVELOPMENT
OF
INFERIOR
COLLICULUS
A
1211
l-2
X
80
70
I
n
1
I
1
1
,
,,,,I
I
1
I
74-29-08
74-29-06
”
1 0’1
FIG.
(74-29)
’
1
1111 ok
’
1
1
210 FREQUENCY
2. Tuning curves for units in 6-day (74-61) and (B). Note that the ordinate for graph A is expanded
paper (Fig. 8 of ref 1) and illustrated in the histogram of Fig. 3. Inspection of this histogram reveals that nearly 70% of units in the inferior colliculus of the kitten had Q factors below 3, compared with less than 30% in
T
I
Go '
7.0
1 I
Id0
I
20'0
'3dO
(kHz)
1 l-day (74-66) kittens (A) and in a 14-day kitten compared with that for graph B. Central nucleus.
the adult. This difference is likely to be composed of two factors. First, as individual tuning curves suggested, the majority of tuning curves were flatter in the neonatal cats than in the adults. Second, units sensitive to high-frequency stimuli
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L. M.
1212
q q
[0,3)
[3,6 )
AITKIN
AND
tween different age groups, a clear tendency for thresholds to decrease from 6 days to reach low levels at 2 1 and 28 days was associated with little dependence on best frequency between 500 and 20,000 Hz. These data were pooled for each age group, converted from decibel values to dynes per square centimeter, means and standard deviations were determined and reconverted to decibel values, and the results were plotted in Fig. 5. Thresholds at 6 and 7 days of age were based on measurements of only three tuning curves but have been included since they are similar in magnitude to the means for the 12 tuning curves at day Il. After this date, a precipitous drop in mean threshold occurred which stabilized near the adult value at day 21. Adult values were computed from material included in the preceding paper (1).
ADULT KITTEN
1
[6,9)
[9,12)
VALUE
OF
[l&15)
'15
0
FIG. 3. Histogram of Q values for tuning curves from the central nucleus of the inferior colliculus in adult cats and kittens. N = 3 1 for kittens, N = 92 for adults.
were less common the adult.
in the kitten
than
D. R. MOORE
in
Thresholds
Tonotopic
The most marked change in the measured parameters as a function of age was in the thresholds of units at their best frequencies. These are plotted for six kittens as a function of best frequency in Fig. 4. Although there is considerable overlap be-
Penetrations through the inferior colliculus of kittens began with the recording of units and background activity which were broadly and irregularly tuned. This pattern persisted throughout the penetration with 6- and 7-day kittens, but was re-
$20-u
organization
-
ZlOO-
z w
0
-
0 m
2 w 8~ f
m
0
l 0
-
Icn W
60-
I-
0;
m
l
0
‘3
0 o
0
0 0
_
I
0.1
I
I~IIII~
r
0.5
1.0
1
2.0 BEST
I
7
A
1’1111
5.0 FREQUENCY
FIG. 4. Scatter plot of best-frequency thresholds of units in six kittens days, w = 11 days, l = 14 days, o = 17 days, A = 21 days, A = 28 days.
.
as a function
0
1
100 (kHz)
I
2oa
of frequency.
300 q
= 6
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DEVELOPMENT
0
FIG.
standard groups
4
5.
8
12
16
AGE
(DAYS)
20
Mean threshold deviations, plotted of cats. -
OF
24
28
INFERIOR
COLLICULUS
1213
placed by the occurrence of units with best frequencies which became progressively higher as the electrode penetrated further, in kittens older than 11 days. The sequences of units with increasing best frequencies obtained in penetrations from ll-, 14-, 17-, and 21-day kittens are plotted in Fig. 6 as a function of relative location of units within a given penetration in 0.5-mm steps. The start of each sequence has been arbitrarily located on the ordinate only for clarity of presentation. Each penetration traversed the entire dorsoventral extent of the central nucleus. A number of features of tonotopic organization are apparent in this illustration. First, for lower frequencies the trends exhibited for each cat were similar and the 3 octaves from 500 to 4,000 Hz were represented in the first 1.2-1.5 mm of the central nucleus. Second, both the number of observations with best frequencies in the 3 octaves above 4,000 Hz and the upper frequency limit increased
ADULT
in decibels SPL, with against age for eight - age-
A
A A
A
0
0
11 DAY
0 0
0 0
0
0
14 DAY 0
0 0
8 o
0
17
0
DAY
0
X It X
21
DAY X x
I
0.3
’
I”“1
0.5
I
1.0
I
2.0 3.0 FREQUENCY
’
I”“1
5-o (kHz)
10.0
x
1
1
2o”o
30.0
FIG. 6. Best frequency in kilohertz (abscissa) as a function of depth from the penetration point at which a tonotopic sequence was first encountered, in 0.5-mm steps (ordinate). The sequence for each cat has been commenced at a different point on the ordinate for clarity of presentation only. Each penetration traversed the entire dorsoventral extent of the central nucleus.
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L. M.
1214
AITKIN
AND
with age. Although it was not quantified, we also noted that the anatomical size of the inferior colliculus increased in this period. Third, with the 17- and 21-day kittens, a number of units were encountered with best frequencies deviating from the trend for the majority of observations. The latter finding could be explained if or fibers were - occasionstellate neurons ally encountered, but it could also be indicdevelop ment of the ative of continual organization of the central nucleus. DISCUSSION
The results presented suggest that the inferior colliculus in the neonatal cat, alto auditory stimuli though responsive soon after birth (10, 16), undergoes substantial maturation in the subsequent 34 wk of life. It would appear that -neuronal thresholds improve from a mean of near 100 dB SPL to a value of about 40 dB SPL 2 wk and over a period of approximately to highthat, concom itan tly , responses more frequency tonal stimuli become common’ although, in view of the small sample size, some caution is needed in vieiing this latter observation. It would bealso seem that an orderly relationship tween the best frequency of a neuron and its location in the centr #al nucleus exists as soon as neurons show selectivitv in their response to the frequency of tonal stimuli. to those of These results are similar from neurons Konishi (11) who recorded in the cochlear nuclei of duck embryos. His study indicated that high bestfrequency neurons appeared after ‘units responding to low frequencies were recorded, that thresholds improved with increasing developmental age, and that tonotopic organization was present at an early age. Relationship
to cochlear
maturation
Some of these results in the cat have been hinted at in previous studies, particularly in those of Pujol (16, 17) and of Maruseva and Malysheva (13). The limited frequency-response ranges of units in very young cats have been found by Pujol to extend from 250 to 2,500 Hz in 2- to S-day-old kittens to more adult values in cats beyond the first week of age (16, 17). It is not clear whether the increase in fre-
D. R. MOORE
quency range is due to cochlear development or to neural maturation, since there has been no equivalent study performed on the development of tuning in cochlear nerve fibers. However, the cochlea shows anatomically adult development 10 days after birth in the cat (18) and cochlear microphonic potentials may be elicited over a wide frequency range in the 6-day rabbit (2). Although such information might suggest that the development of tuning and the diminution of thresholds of inferior colliculus neurons succeed the full development of the cochlea, the recent study of Fernandez and Hinojosa (6) has indicated that the stria vascularis reaches maturity at about the 25th postnatal day and parallels the adult development of the endocochlear potential by the 27-29th day. These times are of a similar order to those for the leveling of response thresholds to adult values in the inferior colliculus. There is little doubt that the neural auditory system of the cat is capable of transmitting some information at birth (12), and unit responses have been detected in the auditory cortex 24 h thereafter (10). However, the elaboration of a tonotopic framework in the inferior colliculus, clear at 11 days, appears to lag behind the structural development of the organ of Corti. It is interesting that many authors (3, 19) have shown that, morphologically, cochlear development proceeds from base to apex. This would appear to contradict our observations that initial evoked activity in inferior colliculus is tuned to a frequency range which would correspond to the apical and middle regions of the organ of Corti. Furthermore, behavioral responses are first initiated to low-frequency tones and are induced by higher tones as much as 4 days after low-tone responses (7). A similar picture exists for the development of brain stem-evoked reponses in the chicken, which show a systematic increase in sensitivity toward higher frequencies as a function of age (23), as do single units in the duck embryo (11). Our results are similar to these, and indicate that unit restimulation sponses to high-frequency (20-30 kHz) become more common in kittens 34 wk in age and, at the same time, the inferior colliculus increases in size.
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DEVELOPMENT
OF
INFERIOR
This discrepancy between cochlear morphology and auditory physiology might be explained if the functional development of the cochlea proceeded from the transmission of low frequencies to the signaling of higher frequencies at a later age. It is probable that the fluid present in the kitten middle ear for the first week of life would impede the reception of higher frequency sounds by the cochlea. The disappearance of such a middle ear impedance could facilitate the development of the high-frequency responsiveness of the cat auditory system. Neural maturation It is possible that cochlear development may also be in part responsible for the decrease in best-frequency thresholds during the first 2-3 wk of life. However, two neural processes, which have been shown to change after birth, may contribute largely to these threshold alterations. First, axons in the inferior colliculus of neonatal cats are poorly myelinated at birth, and myelination proceeds to the adult stage over the subsequent 34 wk of life (16). Such axons would be expected to exhibit low conduction velocities compared with the adult, leading to longer latencies of response in the inferior colliculus. Latencies of evoked potentials have been shown to decrease with age at several levels of the auditory pathway (16, 17, 21). It is also well known that the timing of unit discharges in the adult inferior colliculus is very precise (8, 22). It is likely that such precise timing would be blurred of slowly conducting in a population axons if it required the convergence of information, contained in impulses carried by several such axons, on single inferior colliculus neurons. A further consequence of asynchronous input to a given cell from a population of slowly conducting axons is likely to be an elevation of the thresholds of the responses of inferior colliculus cells. A second consideration relating to threshold changes is that synaptic density is likely to increase after birth in the inferior colliculus as it does in the cat visual cortex (5) where the main phase of synaptic development is critically dependent on
COLLICULUS
1215
function. It is possible that decreases in response threshold seen after the clearing of the middle ear are due to a stimulation of synaptic development in neural auditory structures by the increasing exposure of the auditory pathway to sounds of significance to the developing cat. That such continual development occurs is indicated behaviorally by the observations that, although the inception of hearing occurs in kittens at 5 days (7), more complex responses to sound such as auditory orientating and auditory following responses may take as many as 16 days longer to develop (15). These acousticomotor reflexes related to sound localization are likely to depend on the adult development of the inferior colliculus (4, 22). SUMMARY
Tuning curves were measured for 65 units in the inferior colliculus of seven anesthetized kittens aged from 6 to 28 days. At 2 days of age the inferior colliculus was divisible into central, pericentral, and external nuclei. Evidence was found for broader tuning curves to occur in the pericentral nucleus compared with the central nucleus, as has been observed in the adult. The middle ear was filled with serous fluid to 6 days, while the external auditory meatus remained collapsed until 10 days. Central nucleus tuning curves in kittens were relatively flat with high thresholds. Best-frequency thresholds diminished from a mean of near 100 dB SPL at 6-l 1 days to near 40 dB in the adult. The marked drop in thresholds between days 11 and 21 led to the adoption of the sharp form of tuning curve common for adults. of the central organization Tonotopic nucleus was clear at day 11. Speculations were advanced about the dependence of central auditory maturations on cochlear development, axon myelination in the auditory pathway, and changes in synaptic density as a function of age. ACKNOWLEDGMENTS
We particularly thank Debbi Spain and Moyra Farrington for secretarial and histological assistance. This study was supported by grants from the Australian Research Grants Committee.
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L. M.
AITKIN
AND
D. R. MOORE
REFERENCES 1. 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. Neurt$hy.siol. 38: 1196-1207, 1975. L. An electrophysiological study of 2. ANGGARD, the development of cochlear function in the rabbit. Acta Oto-Laryngol. Suppl. 203: l-64, 1965. B. J. The Temporal Bone 3. BAST, T. AND ANSON, and the Ear. Springfield, Ill.: Thomas, 1949. P., St. LAURENT, J., AND MENINI, C. H. 4. BUSER, Intervention du colliculus inferieur dans I’elaboration et le controle cortical specifique des decharges clonique au son chez le chat sur chloralose. Exptl. Brain Res. 1: 102-126, 1966. 5. CRAGG, B. G. The development of synapses in cat visual cortex. Invest. Ophthalmol. 11: 377-385, 1972. 6. FERNANDEZ, C. AND HIN~JOSA, R. Postnatal development of endocochlear potential and stria vascularis in the cat. Acta Oto-Laryngol. 78: 173-186, 1974. 7. Foss, I. AND FLOTTORP, G. A comparative study of the development of hearing and vision in various species commonly used in experiments. Acta Oto-Laryngol. 77: 202-214, 1974. 8. HIND, J. E., GOLDBERG, J. M., GREENWOOD, D. D., AND Rose, J. E. Some discharge characteristics of single neurons in the inferior colliculus of the cat. II. Timing of the discharges and observations on binaural stimulation. J. Neurophysiol. 26: 32 l-34 1, 1963. 9. KONIC;, N. AND MARTY, R. On functions and structure of deep layers of immature auditory cortex. J. Physiol., Paris 68: 145-155, 1974. 10. K~NIG, N., PUJOL, R., AND MAR*TY, R. A laminar study of evoked potentials and unit responses in the auditory cortex of the postnatal cat. Brain Res. 36: 469-473, 1972. 11. KONISHI, M. Development of auditory neuronal responses in avian embryos. Proc. Natl. Acad. Sci. U.S. 70: 17954798, 1973. 12. MARTY, R. AND T~IOMAS, J. Reponse electrocorticale a la stimulation du nerf cochleaire chez le
chat nouveau-n&J. Physiol., Paris 55: 165-166, 1963. 13. MARUSEVA, A. M. AND MALYSHEVA, V. I. Effects of acoustic stimuli on unit activity of inferior colliculus in cats. Zh. Evol. Biokhim. Fiziol. 6: 225-234, 1970. Translated in: Neurosci. Transl. 15: 41-49, 1970-1971. 14. MERZENICH, M. M. AND REID, M. D. Representation of the cochlea within the inferior colliculus of the cat. Brain Res. 77: 397-415, 1974. 15. NORTON, T. T. ReEeptive-field properties of superior colliculus cells and development of VISual behavior in kittens. J. Neurqbhysiol. 37: 674690, 1974. 16. PUJOL, R. Maturation postnatale du systeme auditif chex le chat. Etude fonctionelle et structurale (These Doct.). Montpellier: Etat. Sci. Nat., 197 1. of tone burst responses 17. PUJOL, R. Development along the auditory pathway in the cat. Acta Oto-Laryngol. 74: 383-39 1, 1972. 18. PUJOL, R. AND HILDIN~;, D. Anatomy and physiology of the onset of auditory function. Acta Oto-Laryngol. 76: l-10, 1973. 19. PUJOL, R. AND MARTY, R. Postnatal maturation of the cochlea of the cat. J. Camp. Neurol. 139: 115-126, 1970. 20. ROMAND, R., GRANIER, M. R., AND MARTY, R. Developpment postnatal de l’activite provoquee dans I’olive superieure laterale chez le chat par la stimulation sonore. J. Physiol., Paris 66: 303315, 1973. H., AND SANTIB~NEZ, G. 21, ROSE, J. E., ADRIAN, Electrical signs of maturation in the auditory system of the kitten. Acta. Neurol. Latinoam. 3: 133-143, 1957. C. D., AND 22. ROSE, J. E., GROSS, N. B., GEISLER, HIND, J. E. Some neural mechanisms in the inferior colliculus of the cat which may be relevant to localization of a sound source. J. Neurophysiol. 29: 288-3 14, 1966. J. C., COLES, R. B., AND GATES, G. R. 23. SAUNDERS, The development of auditory evoked responses in the cochlea and cochlear nuclei of the chick. Brain Res. 63: 59-74, 1973.
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