Encoding of amplitude-modulated tones by neurons of the inferior colliculus of the kitten

Brain Research, 615 (1993) 199-217 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

199

BRES 18957

Encoding of amplitude-modulated tones by neurons of the inferior colliculus of the kitten John

F. Brugge

a,

Barbara

Blatchley

b and Masaharu

Kudoh

c

a Department of Neurophysiology and Waisman Center on Mental Retardation and Human Development, University of Wisconsin, Madison, WI 53705 (USA), b Department of Psychology, Agnes Scott College, Decatur, GA 30030 (USA) and c Brain Research Institute, Niigata University, Niigata (Japan)

(Accepted 26 January 1993)

Key words: Inferior colliculus; Development; Amplitude modulation; Kitten; Auditory system

Responses of single neurons of the central nucleus of the inferior colliculus (ICC) of kittens 4-43 days of age were studied using sinusoidally amplitude-modulated (AM) tones delivered monaurally or binaurally via sealed and calibrated earphones. The carrier frequency of the AM signal was set to the CF of the neuron. CFs ranged from 2-26 kHz. During the about first 2 weeks of postnatal life, ICC neurons responded to sound with periodic bursts of activity. In response to AM tones, discharges of ICC neurons at all ages studied were phase-locked to the envelope of the modulation waveform over a wide range of stimulus level and modulation depth. A linear relationship, independent of SPL, was found between the average phase of discharge on the modulation cycle and modulation frequency. The slope of the line represents a time delay, which was highly correlated with the first-spike latency to tone onset, and hence with the age of the animal. The mean effective phase of the discharge remained relatively constant with age. There was little systematic change in average phase of discharge with changing stimulus level or modulation depth, although the number of spikes evoked and the temporal pattern of the spikes within a modulation cycle could vary. The sensitivity function relating spike synchrony or spike count to modulation frequency was typically band-pass in nature. The most effective modulation frequency (MEMF) was, on average, 15 Hz, far below that reported for adult cat ICC cells. When AM tones were delivered binaurally, the discharge was a periodic function of the interaural phase difference of the stimulus envelopes. The results indicate that prior to the time the cochlea is able to respond to most environmental sounds, monaural and binaural circuits involving the ICC faithfully transmit information pertaining to amplitude-modulated signals in the rate and timing of their discharges. During the next several weeks, when neural thresholds fall to adult levels, ICC circuits are activated by amplitude modulated sounds at levels encountered in the normal acoustic environment even though they are restricted to modulation frequencies below those encoded by the adult.

INTRODUCTION A t b i r t h the kitten a u d i t o r y system is highly u n d e r developed, a n d for a b o u t the first week acoustic thresholds are of such a m a g n i t u d e that it is unlikely the cochlea is activated by any b u t the most i n t e n s e e n v i r o n m e n t a l s o u n d s 9. N o n e t h e l e s s , d u r i n g this first p o s t n a t a l w e e k kittens exhibit the ability to r e s p o n d behaviorally to loud s o u n d , i n c l u d i n g complex s o u n d s a n d conspecific vocalizations, which are rich in amplit u d e f l u c t u a t i o n s 14'17'53. B e t w e e n the second a n d third weeks after birth, thresholds fall a n d kittens b e g i n to show evidence of o r i e n t i n g to the direction of the s o u n d source. T h e s e b e h a v i o r a l data suggest that during the first few p o s t n a t a l weeks the c e n t r a l auditory system is c a p a b l e of t r a n s m i t t i n g , at least crudely,

i n f o r m a t i o n a b o u t the f e a t u r e s of b o t h simple a n d complex sounds, i n c l u d i n g those acoustic cues involved in s o u n d localization. Electrophysiological studies of the a u d i t o r y n e r v e a n d b r a i n s t e m have shown that to be the case for simple tonal stimuli 9. Presently, n o t h i n g is k n o w n c o n c e r n i n g processing of m o r e n a t u r a l complex s o u n d s which c o n t a i n time-varying f l u c t u a t i o n s in intensity a n d a b r o a d spectrum. A m p l i t u d e fluctuations in m a n y n a t u r a l l y occurring s o u n d s are i n f o r m a t i o n - b e a r i n g e l e m e n t s that may be used widely by a n i m a l s t h r o u g h o u t life for c o m m u n i c a tion a n d spatial o r i e n t a t i o n . I n the cat family, for example, a wide variety of a m p l i t u d e - m o d u l a t e d vocalizations are p r o d u c e d in different social situations inc l u d i n g i n t e r a c t i o n s b e t w e e n a kitten a n d its m o t h e r 39'63. I n f o r m a t i o n p e r t a i n i n g to a m p l i t u d e - m o d u l a t e d

Correspondence: J.F. Brugge, 627 Waisman Center, University of Wisconsin, Madison, WI 53705, USA. Fax: (1) (608) 265-3500.

200 ( A M ) signals is t r a n s m i t t e d to the b r a i n of the adult animal in the rate a n d timing of discharges in the auditory nerve array 3°'33'36'37'44"54'67'73.As a rule in the

time locking to the m o d u l a t i o n envelope, (2) selectivity of m o d u l a t i o n frequency a n d (3) sensitivity to i n t e r a u ral phase shifts of the m o d u l a t i o n envelope.

cat, the cochlea acts as a low-pass filter to A M tones, with a cut-off f r e q u e n c y a r o u n d 630 Hz. Both the

MATERIALS AND METHODS

degree of e n t r a i n m e n t to the m o d u l a t i o n e n v e l o p e a n d the rate of discharge are functions of m o d u l a t i o n depth, modulation level 33.

frequency

and

overall

sound

pressure

N e u r o n s of the cochlear nuclei receiving A M inf o r m a t i o n modify the afferent signal substantially 19-21"22'28'34'37'42'43. Phase-locking to the m o d u l a t i o n envelope is e n h a n c e d in the lateral superior olivary n u c l e u s 31'34. Sensitivity to the frequency of an A M signal of n e u r o n s at successively higher levels of the adult auditory neuroaxis may be described by b a n d - p a s s characteristics; such cells may, thus, act as t u n e d filters to A M signals. W i t h i n the inferior colliculus64 a n d auditory cortex ~'5 cells t u n e d to a best m o d u l a t i o n frequency may be organized in a topographic way. Thus, central auditory n e u r o n s may e n c o d e a m p l i t u d e m o d u l a t i o n s in spike timing, in spike rate a n d in the locus of activation. A d d i t i o n a l i n f o r m a t i o n is gained w h e n an A M signal a p p e a r s at the two ears. U n d e r these c o n d i t i o n s the discharge of a n e u r o n in the central n u c l e u s of the inferior colliculus (ICC) may be highly d e p e n d e n t on the i n t e r a u r a l time relationships of the m o d u l a t i o n envelopes 4"1s'31"32'76, a m e c h a n i s m that may a c c o u n t in part for a listener's ability to localize a high frequency complex sound 26'27'40'41'5°'51. T h e ICC plays a pivotal role in processing acoustic i n f o r m a t i o n arising from a wide variety of sources in

These experiments are part of a series of studies on the development of auditory coding mechanisms~'-9. The methods for sound generation, acoustical calibration, preparation of the animal, singlecell recording and data analysis are the same as those described in a previous paper on the kitten inferior colliculus5. Briefly, kittens were anesthetized with sodium pentobarbital and ketamine hydrochloride and their tracheae cannulated. Glass-insulated platinum-iridium microelectrodes were inserted into the exposed inferior colliculus under visual control via a microdrive mounted on a hydraulicallysealed chamber. Stimuli were synthesized and generated via a digital stimulus system, which was under control of a PDP 11/23 computer. Signals were delivered to one or both ears via a sealed and calibrated sound system. Tones were of 3-s duration, with 5-ms trapezoidal rise/fall times, and were delivered every 5 s for 5-50 repetitions. Our sinusoidally modulated stimulus contained three spectral components, one being the carrier frequency and the other two being sidebands equal to the carrier + the modulation frequency. The amplitudes of the sidebands were 6 dB below that of the carrier at 100% modulation. Modulation began at stimulus onset (sine phase) and continued throughout the duration of the signal. With the high-frequency carriers and low modulation frequencies used in our experiments, it is quite likely that the sidebands of the AM signals fell within the response area of at least some of our neurons. Unfortunately, it was not possible within the contact-time we typically had with a neuron to study a response area in sufficient detail to ascertain possible sideband contributions to the AM response. The carrier and sidebands were typically outside the range of frequencies where phase-locking to those stimulus components might be contributing to the temporal response patterns recorded. Sound pressure level (dB SPL re 20 #PA) was that of the carrier tone. Because our aim was to study the steady-state responses to the AM signals, spikes that occurred earlier than 60 ms after stimulus onset were eliminated from data analysis. The locations of recording sites were verified histologically.

the auditory b r a i n s t e m a n d is especially involved in m e c h a n i s m s of s o u n d localization ~'29. A l t h o u g h not

RESULTS

studied systematically in the kitten, the structure of the 1CC is highly u n d e r d e v e l o p e d at birth a n d reaches adult form some weeks t h e r e a f t e r 1,9,46. F u n c t i o n a l un-

General obserc, ations

d e r d e v e l o p m e n t , expressed by ICC n e u r o n s as long onset latency, b r o a d t u n i n g , a n d i m m a t u r e temporal discharge patterns, is associated with this structural immaturity. O n the o t h e r hand, ICC n e u r o n s of kittens may also exhibit some adult-like discharge properties in response to simple p u r e tones p r e s e n t e d m o n a u r a l l y or b i n a u r a l l y 2,5,47,48. In the e x p e r i m e n t s p r e s e n t e d here we wished to d e t e r m i n e the extent to which I C C n e u rons are capable of e n c o d i n g m o r e complex highfrequency s o u n d s c o n t a i n i n g time-varying fluctuations in intensity d u r i n g those few postnatal weeks w h e n the a n i m a l is first experiencing the auditory world a n d w h e n the auditory system is u n d e r g o i n g d r a m a t i c structural change. W e focused our a t t e n t i o n on three p o t e n tial coding m e c h a n i s m s o p e r a t i n g at high stimulus frequencies that are exhibited by adult ICC n e u r o n s : (1)

T h r e e h u n d r e d a n d seven n e u r o n s were isolated in the ICC of 25 kittens r a n g i n g in age from 4 to 43 days. R e s p o n s e s were studied both to m o n a u r a l a n d b i n a u r a l stimulation. M o n a u r a l stimuli were usually p r e s e n t e d at the ear c o n t r a l a t e r a l to the I C C u n d e r study. Typically in both kitten a n d adult cat, s t i m u l a t i o n of o n e ear (usually the c o n t r a l a t e r a l one) with a C F tone evokes an excitatory r e s p o n s e in an ICC n e u r o n . Stimu l a t i o n of the opposite ear alone is either inhibitory (EI), excitatory ( E E ) or w i t h o u t d e m o n s t r a b l e effect (EO), a n d b i n a u r a l i n t e r a c t i o n s are c o m m o n l y exhibited w h e n tones are delivered to both ears simultaneously 1'e9. Eighty-two p e r c e n t (252) of n e u r o n s isolated r e s p o n d e d to p u r e tones, although in the y o u n g e s t a n i m a l s thresholds could exceed 100 dB SPL. Characteristic frequencies (CFs) of these n e u r o n s r a n g e d from 0.1 to 30 kHz. Some of the u n r e s p o n s i v e n e u r o n s were isolated shortly after the electrode e n t e r e d the IC, a n d

201 may have been located just dorsal to the ICC in what is referred to as the pericentral nucleus or dorsal cortex; others were clearly within the ICC and among neurons responding to tones. One hundred forty one neurons that responded to pure tones were tested for their ability to discharge in a phase-locked way to the envelope of an AM signal having a carrier frequency at CF and a modulation frequency between 2 and 100 Hz. Seventy-one percent of those so tested were AM responsive. While recording from many AM-insensitive neurons in kittens as young as 5 days of age, we noted that the unresolved, but audible, background neural activity was phase-locked to the AM envelope. Results presented in this paper were derived from 63 well-isolated neurons with CFs above 2 kHz for which we have substantial parameteric data. All were located within the ICC. Seventy-nine percent of these were from kittens in the age range of 4-21 days; 93% were from kittens younger than 29 days. We concentrated on the first 3 - 4 weeks of postnatal life for it is during this time period that acoustic thresholds fall from a level greater than 100 dB SPL to those of the adult cat. It is also the time during which the cochlea and auditory nerve achieve adult function and when the brainstem is still undergoing dramatic structural and functional change. One animal was studied at 43 days of age, which is when the auditory periphery and brainstem are both close to having adult form and function 9'46. All data were collected with the carrier tone at or near the neuron's CF, which ranged from 2 to 26 kHz. Although we describe some aspects of the pure-tone temporal discharge pattern of developing ICC neurons, we did not study this property in sufficient detail to allow us to correlate it with AM sensitivity 21. With few exceptions (see e.g. Fig. 1), beyond the first several hundred milliseconds of stimulation discharge rate in response to the CF carrier tone alone was not sustained, or was sustained at a relatively low level.

Temporal modulation of the discharge The discharges of young ICC neurons were, as a rule, deeply modulated in response to monaural AM tones within a restricted range of modulation frequencies. We begin with data from a 43-day-old kitten, the oldest animal in the series, for it illustrates the robust AM-sensitivity exhibited by an ICC neuron in an animal whose auditory periphery and brainstem are nearly adult in both form and function. Fig. 1A shows the temporal discharge pattern that resulted from a CF carrier tone delivered to the contralateral ear alone at 24 dB. The cell responded in a vigorous and sustained manner throughout the 3 s of stimulation. This was

Age: 43 Days Carrier Frequency: 7.5 kHz Contralateral Ear Alone: 24 dB Modulation Depth: 0% 40

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2000

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Time (msec)

Modulation Depth: 99% Modulation Frequency: 2 Hz

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20o0 30o0 400o 5000 Time (msee) Fig. 1. A: response to carrier tone alone at CF (7.5 kHz) delivered to contralateral ear at 24 dB SPL. B: response to AM tone; modulation frequency: 2 Hz; carrier frequency: 7.5 kHz; modulation depth: 99%; age: 43 days. On this and subsequent figures: n, number of spikes evoked during tone; r, vector strength; o, average phase of discharge within modulation cycle; tones are 3 s in duration and repeated every 5 s, and the carrier is equal to the CF of the neuron.

followed by a brief pause in firing followed by a resumption of spontaneous activity. Fig. 1B shows the response of that neuron to the same CF carrier tone deeply modulated sinusoidally at a frequency of 2 Hz, which is near the most effective modulation frequency for this cell (see below). Under this condition the discharge was securely time locked to the modulation envelope as evidenced by the six evenly spaced peaks in the peristimulus time histogram corresponding to the six cycles of modulation that occurred during the 3 s of stimulation. The insets in Fig. 1 are period histograms whose abscissae are equal to one cycle of the 2-Hz modulation cycle. The average preferred phase angle (O), expressed as a fraction of the cycle, is given on the figure along with the vector strength (r), a measure of the degree to which the spikes are time locked to the stimulus c y c l e 4'23'33. m value of 1 indicates perfect phase synchrony with all spikes falling within the same histogram bin, whereas a value of 0 implies that the spikes show no preference for their firing position within the modulation cycle. The origin of the abscissa on these and other period histograms in this paper coincides with 0 degrees of phase for a sine wave of modulation.

202 In Fig. 1A the period histogram shows that spikes were essentially randomly distributed over the stimulus cycle (r = 0.04), which is expected since the stimulus contained only the unmodulated carrier tone. The period histogram in Fig. 1B shows that when the carrier was amplitude-modulated a clear preference existed for discharges to occur during a portion of the modulation cycle and around a certain phase on the modulation envelope. We also note in Fig. 1 that the number of spikes evoked by the 7.5-kHz carrier tone alone remained essentially unchanged when a 2-Hz amplitude modulation was imposed. The discharge rate during the excitatory portion of each stimulus cycle exceeds that of the steady state discharge to the carrier tone alone, and spontaneous activity, which is evident during the 2-s off period, is absent during the briefer non-excitatory part of the periodic signal. Whereas the discharges of all ICC neurons studied were entrained to the modulation waveform, the timing of the discharges and the number of spikes evoked varied with changes in the rate and depth of modulation and with overall sound pressure level.

A

Effects of changing modulation frequency on rate and timing of discharges At sound pressure levels some 20-30 dB above threshold and at modulation depths of 90-99%, the number of spikes evoked during the 3-s stimulus was a function of modulation frequency (fm) for all neurons at all ages studied. In Fig. 2, the number of spikes evoked by repeated 3-s AM signals delivered to the contralateral ear alone as function of modulation frequency is plotted for 6 neurons at either end of our age spectrum. These results are representative of our larger data set. Because we are interested here in change in sensitivity and not in spike count per se, the spike count has been normalized. For two cells at 43 days of age (Fig. 2A, units 2 and 4) the functions are sharply peaked at 2 Hz and 30 Hz, respectively, whereas for the third (unit 7) the curve exhibits a broad maximum between about 60 and 100 Hz. We refer to the peak in the spike count-vs-fm function as the 'most effective modulation frequency' (MEMF) for that neuron. Below, in Fig. 2B, the corresponding curves relating synchrony (to the modulation cycle) and modulation fre-

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M o d u l a t i o n F r e q u e n c y (Hz) Fig. 2. Sensitivity to modulation frequency at 43 days of age (A,B) and 4-5 days of age (C,D) expressed as a change in spike count (normalized) and discharge synchrony to the modulation envelope.

203 quency are shown. In all three cases, the discharges are solidly locked to the modulation cycle at and on either side of the MEMF. For one of the cells (unit 7), phase-locking is in evidence at modulation frequencies exceeding 100 Hz. Fig. 2C,D illustrates similarly plotted spike count and synchrony data for three neurons from three kittens aged 4 - 5 days. The forms of the functions are similar to those shown for the 43-day-old animal. Usually the shape of the spike count-vS-fm curve is not identical to that of the synchrony-vsofm function nor are the peaks in the two functions often in exact register. For most neurons locking of the discharge to a particular portion of the modulation cycle extended over much of the modulation frequency range to which the neuron responded by changing its discharge rate. Several other curves are shown in Fig. 9 for neurons in kittens of 15-22 days of age. For two cells shown here (Fig. 9A, D) spike count-vs-fm functions are similar to those illustrated in Fig. 2. The curves exhibit a single maximum even at modulation frequencies covering several hundred Hz (not shown). For two other cells illustrated in Fig. 9B,C, the countvS-fm functions show somewhat unusual characteristics; discharge rate first rose to a maximum with increasing modulation frequency then fell to a minimum only to rise again at successively higher modulation frequencies. In Fig. 9A, D phase-locking was high at modulation frequencies as low as 1 or 2 Hz, and remained high over the full range of modulation frequencies that excited the neuron, whereas quite the opposite was seen in Fig. 9C where phase synchrony fell precipitously above about 20 Hz. The most effective modulation frequency ranged from 1 or 2 Hz to 120 Hz (Fig. 3). Ninety percent of the neurons in our sample showed a peak of sensitivity below 50 Hz; MEMFs above 50 Hz were not seen in animals younger than 10 days of age. The median peak value for the 50 curves (from 40 neurons) was 15 Hz, which is considerably below that reported in the adult cat ICC by Langner and Schreiner 38. Moreover, there was no significant increase in the M E M F with age, at least up 3 - 4 weeks postpartum. Thus, whereas ICC neurons in kitten and adult cat are tuned to a particular modulation frequency or range of modulation frequencies, the adult upper limit of modulation-frequency detection apparently is not reached in the first several postnatal weeks.

Average discharge phase vs. modulation frequency When stimulus level and modulation depth were held constant, the average phase of the discharge shifted systematically as a function of modulation fiequency. Fig. 4 illustrates this relationship with a series

25

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Age

(Days)

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Fig. 3. A: distribution of the 'most effective modulation frequency', defined as the modulation frequency that evoked the largest number of spikes. Values obtained at different SPLs for the same neuron are included. B: relationship between the 'most effective modulation frequency' and postnatal age. Duplicate values plotted as single points. Regression line determined for MEMFs below 90 Hz.

of period histograms derived from the responses of a 22-day-old ICC neuron to AM tones having modulation frequencies between 2 and 200 Hz. Whereas the discharge was strongly locked to the modulation envelope at all modulation frequencies illustrated, below about 30 Hz the spike distribution within each cycle was temporally complex (see also Figs. 7 and 8). A plot of the average phase of the discharge versus modulation frequency is illustrated in Fig. 5 for those 33 neurons for which adequate data were available. Here the average phase was derived only from those responses for which the period histogram indicated a near-normal distribution of phase-locked spikes (see Greenwood 24 and Joris and Yin 33). The data are grouped by age over the range of ages studied. For each cell, the data points are fitted by a straight line; correlation coefficients for the 33 curves exceed 0.90. As for phase-locked responses to low-frequency tones 3, we interpret the slope of each function as representing a time delay between when the electrical signal appears at the earphone and when the electrode records the resulting evoked action potentials; the time includes small acoustic and middle ear delays, cochlear transmission time, and a neural delay from the cochlea to the ICC25'33'57. The linear relationship indicates that this time delay is independent of modulation rate. There is a positive correlation between the onset latency to the carrier tone, which is age dependent 5, and the time delay computed from the phase-vs-frequency plots (Fig. 6). This can also be seen in the somewhat

204

Age: 22 Days Carrier Freq: 2400 Hz C: 34 dB N=487 0 = o.2o r= 0.44

N=497

O = 0.27 r = 0.57

N=450 O = 0.35 r= 0.60

N=486 0 = 0.45 r = 0.88

steeper slopes of the phase-vs-frequency plots obtained at 4 - 2 0 days of age as compared to those obtained in older kittens. For 84% of neurons studied this way the Y-intercepts of the regression lines varied between about + 94 and - 4 8 degrees, with a median value of 75 degrees. We refer to this average phase derived from the Y-intercept as the 'average (or mean) effective phase', which we take to reflect the average phase on the modulation envelope around which spike discharge occurs. For most neurons, the values obtained indicate that the neuron responded to the rising phase of the stimulus envelope. For one (Fig. 5, 22 days), however, the intercept occurred much later in the stimulus cycle where amplitude was falling. This was a neuron that responded only to the termination of a pure-tone stimulus. Unlike the slope of the regression line, the distribution of the average effective phase did not change with advancing age.

Effects of changing modulation depth N = 576 0 = 0.50 r = 0.93

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Most of our data were derived from responses of ICC neurons to AM tones modulated at depths of 90-99%. In a smaller sample of cells (n---20) we studied sensitivity to changes in modulation depth. These experiments were carried out at or near the most effective modulation frequency for the cell. In all kittens studied between 4 and 43 days of age, the results were similar. Fig. 7 illustrates this in period histogram form for five ICC neurons 4-22 days of age. Shown here are the changes in temporal discharge pattern, time locking and phase angle of the discharge and spike count that occurred as modulation depth was changed while modulation frequency and SPL remained constant. Stimulus level was held close to the cell's threshold to the carrier tone alone. Modulation depth is shown to the right of the histograms. In each case, phase-locking to the stimulus cycle was exhibited at modulation depths as shallow as 10%. As modulation deepened the number of spikes evoked during the stimulus (n) increased and the distribution of spikes within the excitatory region of the modulation cycle became more restricted. In some cases the spike distribution also became complex, as shown in Fig. 7 where at 4 days the histogram splits in two at modulation depths greater than about 40%. Whereas the numbers of spikes and temporal patterns could change substantially with changing modulation depth, there was little or no systematic shift in the average phase of the overall response regardless of age, and if there was it occurred near the threshold for

Fig. 4. Period histograms showing the shift in phase of the discharge as a function of modulation frequency.

205 discharge modulation. We do not know the extent to which a phase shift might occur at modulation frequencies distant from the most effective one.

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Neurons in 10 kittens ranging in age from 4 to 21 days were studied over a relatively wide range of SPLs. As a rule, over a cell's dynamic range the spikes were locked to the modulation cycle, although as stimulus level was raised there was a change in the temporal pattern of the discharge within a stimulus cycle. Fig. 8 illustrates results from three ICC neurons in kittens 4, 7 and 20 days of age. It was commonly seen that with increasing level there was a spread in the distribution of spikes. For two ceils shown here the distribution split to form complex discharge patterns within a modulation cycle. Peak splitting was seen usually at the lowest modulation frequencies and was not confined to the youngest kittens; indeed it has been reported for ICC cells of adult cats 38"49. For complex response patterns, it was difficult to assign a preferred phase of firing 33. Nonetheless, taking into account the discharge pattern as a whole, from inspection of the histograms there was little shift in the preferred region of the modulation cycle over most of the dynamic range of the ceils. Fig. 9 illustrates for four neurons AM sensitivity at different modulation frequencies and sound pressure levels. Over a range of 10-50 dB the most effective modulation frequency remained relatively constant. The functions relating synchrony to modulation frequency behaved quite similarly over that stimulus range as well. For clarity the phase-vs-frequency plots in this figure were shifted upward to be centered on the graph. Phase-vs-fm plots were linear regardless of sound pressure level. The slopes of the functions varied little, although for the cell illustrated in Fig. 9D one curve departs substantially from others in the series. Except for the cell shown in Fig. 9B, the functions did not superimpose, although the Y-intercepts differed by no more than about 0.5 cycle (Fig 9C).

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In addition to the relatively low temporal resolving power exhibited by young ICC neurons as compared to their adult counterparts, there were also signs of functional underdevelopment in the time patterns of the

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Fig. 5. Cumulative average phase of the discharge during the modulation period as a function of modulation frequency for 33 neurons grouped according to postnatal age• Data points were fit with a straight line.

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discharge of cells in the youngest animals in the series (Figs. 10 and 11). Fig. 10 illustrates the discharge pattern of three ICC cells in kittens 4 - 7 days of age at different stimulus levels. In each case the stimulus was an unmodulated pure tone of 500-ms duration at the neuron's CF. The most obvious feature of the discharge was its periodic nature; the temporal separation of the peristimulus bursts of activity was between about 100-150 ms, and depended on stimulus level. By the end of the second postnatal week the evoked bursts are no longer in evidence. The evoked bursts described above interact with the temporal rhythm of spikes imposed by an AM signal. Fig. 11 illustrates this with data from a 5-day-old kitten. The stimulus was a 3-s AM signal having a carrier frequency equal to the neuron's CF. Stimulus level was held at 114 dB, which is within about 20 dB of threshold for a kitten of this age 9. Modulation frequency was the relevant parameter. For each modulation frequency, from 0.3 to 100 Hz, the data are presented in three forms: dot raster (top), peristimulus time histogram (middle) and period histogram binned on the period of the modulation cycle (bottom). Over the range of modulation frequencies illustrated, the discharges are locked to the modulation envelope, as shown by the single major peak in each period histogram. At the lowest frequencies (0.3 to 1 Hz), the spikes are distributed with respect to both the period of the modulation envelope and the periodicity of the evoked bursts. This is best seen in the dot rasters and PSTHs where the discharge pattern locked to the stimulus envelope includes spike bursts of about 100-150 ms separation. At 2 Hz the evoked bursts are still recognizable, but at 5 Hz the discharge timing is determined largely, if not entirely, by the amplitude modulation. At still higher modulation frequencies (e.g. 30-100

Binaural interactions We have so far only described the sensitivity of kitten ICC neurons to sinusoidally modulated signals delivered to one ear alone. A high proportion of ICC neurons in the adult animal are sensitive to interaural phase or level differences of dichotically presented tonesl'29; some are sensitive to the interaural phase differences of amplitude-modulated tones 4'3a'32'76. We also know that ICC neurons in kittens of the ages studied in this series of experiments are sensitive to small interaural intensity differences 2'5'46'47 and, for cells responding to low frequencies, to small interaural phase differences 5. We now report that kitten ICC cells excited by high frequencies are equally sensitive to small interaural time differences in the envelope of the sinusoidally amplitude-modulated tone. We studied this sensitivity using two stimulus paradigms. In the first, 3-s AM tones having the same high carrier frequency and low modulation frequency were delivered to each ear. The phase of the modulation envelope of the signal to one ear was then shifted in steps of 30 degrees, with data being collected at each of the stationary 30-degree steps. The phase of the carrier signal remained constant. The carrier frequency was above 3 kHz and, thus, not capable of evoking its own phase-locked activity at these ages 1°'35. The results of such an experiment in three animals, age 5, 13, and 22 days, are shown in Fig. 12. The neurons could be placed in the EI binaural category, that is, they were excited by stimulation of the contralateral ear and inhibited by stimulation of the ipsilateral ear x'29. In Fig. 12 spike count is plotted against the interaural phase of the modulation envelope for one complete modulation cycle. The functions are cyclic indicating that the neurons each responded to the phase difference of the modulation envelopes at the two ears. Two other features of the curves are noteworthy. First, regardless of the modulation depth (Fig. 12A) or frequency (Fig. 12B,C), the peaks of the curves tend to coincide for these neurons. If true for the whole population, these data suggest that kitten ICC neurons not only are sensitive to IPDs of an AM envelope, but may also exhibit a possible 'characteristic delay' for AM signals with high carrier frequency. Second, the number of spikes evoked by the most favorable interaural time delay is not greater than the number evoked by stimulation of the excitatory ear alone. In other words, the effect of shifting interaural phase was to reduce the number of spikes evoked by the excitatory ear. This observation suggests that inter-

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10 Hz modulation frequency at the contralateral, excitatory ear. There is no evidence for l l - H z modulation from the ipsilateral ear despite the fact that the input from this ear was a powerful and phase-locked (albeit inhibitory) one. A final observation is that the upper limit for locking to the modulation envelope, 250 Hz, is the highest recorded in our sample, and while we have no further systematic data on this upper limit when binaural stimulation is employed, it may be the case that interaural phase sensitivity extends the range of phase-locking to amplitude-modulated signals.

We have found that within the first weeks of postnatal life, even prior to the time the animal experiences normal environmental sounds, neurons of the ICC of kittens are capable of encoding amplitude-modulated high-frequency tones in both the rate and timing of their discharges. Although these coding mechanisms are operating during this early postnatal period, the upper limit of modulation frequency that can be detected is considerably below that detected by ICC neurons in adult animals• The fact that kitten ICC neurons respond in a time-locked way to fluctuations in stimulus intensity may be predicted. During the early postnatal period auditory nerve fibers and neurons of the cochlear nuclei and 1CC exhibit sensitivity to changes in sound pressure level of CF tones that is as high as, if not higher than, that of their adult counterparts 5'11'35'61• In addition, kitten ICC neurons show adult-like sensitivity to differences in interaural phase 5 and interaural intensity2'5'47'48 of low- and high-frequency tones, respectively. In general, the synchronized response of a kitten ICC neuron to a monaural sinusoidal AM signal does not reproduce accurately the waveform of the modulation envelope. Depending on modulation parameters, the temporal pattern within a modulation cycle may be highly restricted or may spread well beyond half the period of the waveform, and it may show two or more peaks of activity. Spontaneous activity may be suppressed during the non-excitatory portion of the modulation cycle. These temporal response patterns, and consequent distortion of the period histogram, are not confined to the young kitten. They have been recorded in adult animals, using various AM signals, from auditory n e r v e f i b e r s 3°'33'73 and neurons of the cochlear nuclei 19'28, superior olivary complex 31 and inferior colI i c u l u s 4'38'49'57'59. In our sound system, harmonic distortion is some 40-50 dB below that of the fundamental and there was no visible evidence of a distorted signal waveform at the tympanic m e m b r a n e as recorded with a probe microphone. Several mechanisms in the cochlea and central nervous system may be at play in shaping these temporal response patterns in the ICC of both kitten and adult cat. Distorted period histograms derived from auditory nerve fiber responses to AM signals, which account for the frequently described non-monotonic relationship between synchrony to an AM stimulus and SPL, may be attributed to cochlear saturating non-linearities reflected in rate-vs-level functions 66. Similar processes may be operating in the young kitten where the rate-

213 a train of sound bursts, we should expect to find in the discharges of kitten ICC cells some variety of temporal response patterns within a modulation envelope similar to those exhibited by their adult counterparts 5. Although the AM response properties reported here resemble in many ways those recorded in the adult ICC under similar stimulus conditions, they also exhibit differences which are associated with highly underdeveloped peripheral and central auditory systems. During the first week or two of postnatal life, the discharge of an ICC neuron contains a rhythmic pattern of toneevoked bursts of spikes 55'56. These bursts probably do not originate in the ICC, for they are also seen in the discharge patterns of young auditory nerve fibers 13'7° and cochlear nuclear neurons tz'62'7°'71. Nor do they have the characterisics of 'intrinsic oscillations' described by Langner and Schreiner 38 in the adult ICC. The fact that they appear to superimpose on the tem-

vs-level functions of auditory nerve fibers obtained with steady-state tones are similar in shape if not steeper than those of the adult 35'61. More complex mechanisms may be engaged in the cochlear nuclei where, in the adult, AM responses are obtained over an operating range that may exceed that of auditory n e r v e fibers 21'22'43'67. The temporal patterning of discharges of adult brainstem auditory neurons to pure-tone bursts involves both the cells' intrinsic membrane properties as well as cell-cell interactions of excitation and inhibition 52'6°'77. These mechanisms are manifest early in life in the cochlear nuclei 12'62'7°-72 and inferior colliculus5. The results of Frisina et al. 21'22 in the adult gerbil suggest a relationship between the mechanisms generating these tone-evoked temporal discharge patterns and AM selectivity of cochlear nuclear neurons. If we consider a sinusoidally amplitude modulation signal as

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(msec)

Fig. 13. Sensitivity, expressed as phase-locking to the modulation envelope, to an AM beat created by delivering 10 Hz AM tone to one ear and an 11 Hz AM tone to the other. Abscissa for the peristimulus histogram is 3 s, which is the length of the modulated signal.

214 poral rhythms imposed by AM tones suggests that the mechanisms that generate them are quite separate from the ones that generate the phase-locked response. In the adult animal, from cochlear nuclei to auditory cortex, the function-relating spike count or synchrony to modulation frequency is typically band-pass in nature. A certain modulation frequency is enhanced over all others and hence such a neuron may be said to be 'tuned' to this preferred modulation frequency. The shape of the modulation transfer function may vary from one neuron to the next, an observation also made in the adult cat by Langner and Schreiner 38 under similar experimental conditions. Collicular neurons in the kitten exhibit a similar property, although the most effective modulation frequency is far below that obtained in the adult. Our observations in young kittens place the average most effective frequency for spike rate at 15 Hz; no more than 10% of our sample have MEMFs above 50 Hz. While for any given cell the shape of the synchrony-vs-fm function may differ from that of the spike-count curve, the fm most effective for phase-locking is not dissimilar to that for spike rate, which is also the case in the adult ICC 3s. Langner and Schreiner 3s found that in the adult cat ICC the peak rate sensitivity to 100% sinusoidally amplitude-modulated tones (referred to as the 'best modulation frequency', or BMF) ranged between 10 Hz and 1000 Hz, with most of the BMFs falling between 30 and 100 Hz. In the adult rat, Rees and M¢ller 58 reported that ICC neurons were tuned to modulation frequencies between about 80 and 120 Hz, and the observations of Rees and Palmer 59 in the guinea pig are in close agreement with figures for the rat and cat. Thus, whereas the shapes of the modulation transfer functions show no clear age-related differences, the distributions of most effective modulation frequencies of adult animals and kitten are barely overlapping. Schreiner and Langner 64 reported that in the adult cat BMFs were distributed in the ICC in a topographic way. Neurons having the highest BMFs were clustered near the center of the ICC, and progressively lower ones were recorded at increasing distances thereby forming iso-BMF contours. We did not attempt to map the kitten ICC for such organization, but our electrodes did frequently penetrate much of the central region of the nucleus where in the adult the highest BMFs would be expected to be found. Thus, unless the topographic organization shown by Schreiner and Langner 64 for the adult cat is drastically different in the kitten, it is unlikely that the relatively low peak frequencies exhibited by kitten ICC cells was due to an electrode-sampling bias. Rather, the temporal resolving power of young ICC cells, as expressed by rate and

synchrony sensitivity to changing modulation frequency, appears to be considerably below that of adult ICC neurons. We found no strong age-related changes in the peak frequency during the first 6 weeks of life, which suggests that a shift to higher MEMFs occurs quite late. Eggermont t6 in his study of auditory cortical development came to the same conclusion, that periodicity coding takes considerably longer to mature than do some other response properties. Based on their finding of a positive correlation between AM sensitivity and tone-evoked discharge patterns in the gerbil ventral cochlear nucleus, Frisina et al. 21'22 postulated several mechanisms by which lowpass characteristics exhibited by auditory nerve fibers could be transformed to band-pass properties in the cochlear nuclei (and presumably in more successive central auditory nuclei). These include off-CF inhibition, patterns of excitatory and inhibitory afferent inputs, intrinsic membrane properties, and input from more central auditory nuclei. Cochlear nuclear neurons of kittens exhibit tone-evoked discharge patterns and tonal response areas that have many of the properties of their adult counterparts 8't2"62'7°'71, thereby revealing at a very early age some of the mechanisms postulated by Frisina et al. 22. If in this regard any or all of these mechanisms come into play in the adult then we might expect them to be operating in the cochlear nuclei of young kittens as well, albeit at a somewhat reduced level of efficacy. Thus, it is not unreasonable to suppose that the band-pass properties of IC cells of kittens and adult cats are, in part at least, reflections of neural mechanisms operating in the cochlear nuclei. Because the distribution of peak modulation sensitivity shifts to lower modulation frequencies at successively higher levels in the auditory pathway, there must also be contributions made by circuits central to the cochlear nuclei. Thus, the relatively low peak sensitivity to AM exhibited by kitten ICC cells is likely due to a combination of underdeveloped mechanisms operating in the ICC and in structures peripheral to it. During the first weeks of life, the onset discharge latency to a pure-tone burst is relatively long for neurons in the cochlear nuclei and becomes progressively longer at successive levels of the central auditory pathway including the ICC 9. Langner and Schreiner 3s report a positive correlation between onset latency and the most effective fm for adult ICC neurons: short latencies are associated with high BMFs. In addition to onset latency, the recovery cycle of a developing auditory neuron may also be prolonged 72. It was found previously that young cochlear nuclear neurons may be limited in their ability to entrain to clicks at high click rates t2. In a more recent systematic study of periodicity

215 coding by kitten auditory cortical neurons, Eggermont 16 found that click-following reached adult levels at a relatively late stage, and he attributed this to the gradual change in the duration of post-activation suppression or hyperpolarization. Thus, onset and recovery times may be key factors in governing the ability of young central auditory neurons to entrain to rapidly changing amplitude fluctuations. Our findings in the kitten ICC of sensitivity to interaural phase differences in the envelope of an amplitude-modulated signal are in close agreement with those of Yin et al. 76 in the adult cat and Batra et al. 4 in the adult rabbit. Although Crow et al. t5 were the first to present evidence to suggest that ICC neurons were sensitive to interaural differences in the timing of the envelope of a high-frequency complex sound, the definitive experiments on this point were carried out by Yin et al. 76 who showed that cat ICC neurons respond to interaural phase differences of the envelopes of amplitude-modulated (trapezoidal) sounds and that this response depends on the preservation of temporal information of the modulating waveform. Later, Batra et al. 4 came to the same conclusion in their studies of ICC neurons in the adult rabbit using sinusoidal AM signals in a paradigm similar to the one we have employed. Both the Yin and Batra groups showed that adult IC neurons also respond to binaural beats of the modulation envelopes 4'76. One striking difference between our kitten results and those of adult cat and rabbit was the comparatively low modulation frequencies at which kitten ICC cells showed IPD sensitivity. This limit is imposed by the neurons' relatively low band-pass characteristics to AM signals. While the highest fm at which IPD sensitivity was shown was around 200 Hz, this was exceptional. This observation is consistent, however, with the data of Batra et al. 4, which suggested that the upper limit for AM IPD sensitivity could be extended with binaural stimulation. Previously we showed that low-frequency ICC neurons in kittens as young as 12 days of age exhibited interaural phase sensitivity 5 similar to that of the adult 75, and it is possible that such sensitivity is present even earlier considering the fact that in the anteroventral cochlear nucleus low-frequency phase-locking is in evidence during the first postnatal week ~°. The upper frequency limit for this temporal coding, like that for AM coding, was also well below that recorded in the adult animal 9. The results of Batra et al. 4 suggest that the mechanisms involved in detecting interaural phase differences of the modulation envelope at high frequency are the same as, or very similar to, those involved in detecting interaural phase differences of

low-frequency tones; the output of a neuron receiving bilateral input phase-locked to the modulation envelope is a function of the timing of spikes arriving over the two monaural pathways. For E1 neurons, the interaction involves phase-locked excitation from one ear and phase-locked inhibition from the other. While the mechanisms for interaural phase sensitivity at high and low frequencies may be the same or very similar, the primary sites in the brainstem where these interactions take place are not likely to be the same. The medial superior olivary nucleus is the site where much of low-frequency tonal IPD sensitivity is established TM, whereas the lateral superior olivary nucleus appears to be the place where high-frequency envelope IPD sensitivity is first encountered 31'32'34, for it is here that cells are especially sensitive to interaural intensity differences. Such a dynamic shift in l i D would occur when there is relative movement between a sound source and the ears. Recent studies of the adult auditory cortex 68'69 showed that neurons in these areas are sensitive to dichotic oppositely directed AM-ramp stimuli that mimic an l i D component of a moving stimulus. Thus, the presence during the first week of postnatal life of sensitivity to the interaural phase of AM signals suggests that the kitten will be capable of detecting some of the more complex, biologically relevant sounds in its natural environment at the time of onset of hearing. Acknowledgements. We wish to thank Ravi Kochhar for computer programming, Bruce Anderson for electronics help and Shirley Hunsaker for photography. This work was supported by NIH Grants HD03352, DC00398 and DC00116.

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