Responses of peripheral auditory neurons to two-tone stimuli during development: I. Correlation with frequency selectivity

HSIRII R[SlmRCH ELSEVIER

Hearing Research 77 (1994) 135-149

Responses of peripheral auditory neurons to two-tone stimuli during development: I. Correlation with frequency selectivity Janet L. Fitzakerley 1, JoAnn McGee, Edward J. Walsh * Department of Physiology, Creighton University, and Boys Town National Research Hospital, 555 North 30th Street, Omaha, NE 68131, USA

(Received 22 June 1993; Revision received 23 February 1994; Accepted 13 March 1994)

Abstract

The responses of peripheral auditory neurons to two-tone stimuli were used to inferentially examine the nature of cochlear processing during development. Rate suppression was not seen in the youngest animals, and was first observed at 77 gestational days, in units exhibiting adultlike frequency selectivity. Suppression was highly correlated with the degree of tuning, and neurons were segregated into three classes based on these responses. Broadly tuned neurons (type I B) with low characteristic frequencies (CFs) did not exhibit suppression, and were observed early in postnatal life. Sharply tuned, but still immature neurons (type I s) exhibited suppression, but to a lesser degree than mature neurons (type M). One interpretation of these results is that basilar membrane mechanics are linear during the final stages of cochlear development, indicating that the immature signal transduction process is fundamentally different from that of adults. Key words: Development; Auditory nerve; Cochlear nuclear complex; Two-tone suppression

I. Introduction

The nonlinear p h e n o m e n o n of two-tone suppression has been described extensively in adult animals, and has been used to make inferences about the nature of signal transduction in the cochlea. U n d e r two-tone conditions, the second tone (i.e., the test tone) is thought to diminish the contribution of an energy-dependent (active) process normally responsible for enhancing basilar m e m b r a n e vibration at the cochlear location generating the response to the first tone (i.e., the probe tone), resulting in a decrease in basilar m e m b r a n e displacement and in neuronal discharge rate (Ruggero et al., 1992; Geisler et al., 1990; C h e a t h a m and Dallos, 1992). The active process is also thought to be responsible for the high degree of frequency selectivity exhibited by peripheral auditory neurons (Neely and Kim, 1983).

* Corresponding author. Fax: (402) 498-6351, Internet: [email protected]. i Present address: Center for Hearing Science, Johns Hopkins University School of Medicine, Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205, USA 0378-5955/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0378-5955(94)00054-T

T h e r e is considerable anatomical evidence that several of the structural properties thought to be necessary for mature cochlear transduction, such as the relationship between the tectorial m e m b r a n e and the outer hair cell stereocilia, as well as the thickness of the basilar m e m b r a n e , undergo dramatic changes during the final stages of inner ear differentiation (Larsell et al., 1944; Lim, 1972; Kraus and Aulbach-Kraus, 1981). In addition, numerous physiological studies have documented changes in tonotopic representation and a lowering of acoustic thresholds during this period of morphological differentiation (Rubel, 1978; Lippe and Rubel, 1985; Walsh and McGee, 1986; Echteler et al., 1989). Threshold vs. frequency (tuning) curves recorded from perinatal kittens are typically broad and do not possess the sharply tuned 'tip' region characteristic of those observed in adults (Romand, 1983; Dolan et al., 1985; Walsh and McGee, 1990). Based on these data, it can be hypothesized that the contribution of the active process required for expression of adultlike nonlinearities is significantly altered during development, and, therefore, that the developing inner ear may not process sound in the same nonlinear manner as the adult. In this and accompanying papers, the nature of cochlear transduction was inferentially studied during develop-

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ment through the use of a two-tone suppression paradigm, in order to assess the linearity of signal processing in the immature peripheral auditory system.

2. Materials and Methods Animals

Data were collected from 70 kittens, ranging in age from 69 to 137 gestational days, and from 13 adult animals, that were older than 240 gestational days (approximately 8 postnatal months) of age. The data collected from these animals were not exclusively directed toward the experiments reported here, thereby inflating the number of subjects required to complete this study. All of the kittens were obtained from an in-house breeding colony, and gestational ages were determined from a 24 hour breeding period. The mean gestational length was 67 + 2 days for the 42 litters which provided animals for this study. Gestational age was used in this investigation to reduce variability that may appear when data are analyzed in terms of postnatal age. The majority of adult cats were obtained from random sources. Animals were housed within the animal facility at Boys Town National Research Hospital (BTNRH). The care and use of all animals were in accordance with the guidelines of the animal welfare committee of Creighton University and BTNRH. This research was supported by NIDCD grants #DC00034 and #DC00982 awarded to EJW. Surgical procedures

Animals were anesthetized with sodium pentobarbital (Nembutal) administered intraperitoneally (IP), and supplemented with ketamine hydrochloride (Ketaset) given intramuscularly (IM). All animals were maintained at a surgical level of anesthesia, with the initial dosage of pentobarbital being adjusted between 25 and 40 mg/kg depending upon age and body weight. Supplemental doses of pentobarbital (2.5-5 mg/kg IP) or ketamine (2.5-5 mg/kg IM) were administered as needed to maintain areflexia. Atropine sulfate was administered prior to surgery (approximately 0.25 mg/kg IM). A tracheotomy was performed to ensure unobstructed breathing, and the animals were ventilated, if necessary, using a small animal respirator (Harvard Apparatus). Body temperature was maintained at approximately 37.5°C using a thermostatically controlled heating blanket (Harvard Apparatus). The right pinna and external auditory canal were resected to the level of the annular ring, allowing visualization of the tympanic membrane. In older animals, a small hole was drilled in the bulla and a small diameter ventilation tube was inserted to allow equalization of the air pressure in the middle ear cavity (Guinan and Peake, 1967). The occipital bone, includ-

ing the region of the posterior fontanelle in younger animals, was exposed by reflecting neck and scalp muscles, and the animals were positioned in a stereotaxic apparatus (David Kopf Instruments). Kittens less than three weeks of age were secured by cementing the skull to a rodent bite bar. Adults and older kittens were held in the stereotaxic apparatus using a modified supporting system which held the skull via standard zygomatic and palate bars and a specialized bar which was placed against the nuchal crest. The posterior fossa was opened and the cochlear nucleus/auditory nerve complex was exposed by aspirating cerebellar tissue. The decision to record from auditory nerve fibers or cell bodies of primary-like neurons in the anteroventral cochlear nucleus (AVCN) was based on the stability of the preparation, as it was not possible to obtain long duration recordings from auditory nerve fibers when brainstem movements were significant. For fiber recordings, the auditory nerve was exposed at its exit from the internal auditory meatus by wedging small pieces of cotton between the brainstem and the temporal bone. Following an overdose of pentobarbital, the animals were sacrificed by transecting the brainstem or by intracardiac perfusion of fixative solutions necessary for a separate anatomical study. Acoustic system and calibration

The sound delivery system consisted of two independent channels. In the majority of experiments (57/83 overall; 43/59 where two-tone data were obtained), one signal was generated by a minicomputer (Digital Equipment Corporation PDP 11/83), and the other signal was produced by an oscillator (Tektronix SG 502). In the remaining experiments, both waveforms were generated by the computer. Digitally synthesized waveforms were transferred to a custom-built DMA waveform generator board (BTNRH), which was interfaced with a Q-bus backplane and controlled by the computer. Clock rates were regulated by a programmable crystal-controlled clock (Syntest) and transferred to a 16-bit digital-to-analog converter (DAC) (Analogic). Both stimuli were amplified with an 80W/channel, low distortion amplifier (Crown International 150A), digitally shaped and gated using custom-built hardware (BTNRH), and passed through an anti-aliasing filter. Tone bursts were symmetrical and linearly ramped. Stimuli were attenuated using custom-built attenuators (BTNRH), which had a range of 127 dB, a resolution of + 1 dB and an accuracy of 1%. Control of the stimulus generation system was accomplished via the PDP 11/83, and was fully integrated with data acquisition. Stimuli were mixed acoustically and delivered to the ear via two dynamic earphones (Beyerdynamic DT-48), which were positioned adjacent to the exposed external auditory meatus using a speculum. A closed acoustic system was established

J.L. Fitzakerley et aL / Hearing Research 77 (1994) 135-149

by sealing the connection with ear mold material (Audalin). Animals were placed within a double-walled sound attenuating chamber (Industrial Acoustics Corporation) for the duration of the experiment. The acoustic system was calibrated in vivo prior to each recording session. Continuous pure-tone signals were generated by the tracking oscillator of a wave analyzer (Hewlett Packard 3581A) and delivered to the earphones. Sound pressure levels were measured at the tympanic m e m b r a n e using a 1 / 2 " condenser microphone (Briiel and Kj~er 4138), which was inserted into a probe-tube assembly coupled to the earphone speculum. Discrete frequencies were sampled in 20 Hz steps between 20 Hz and 5 kHz, and in 100 Hz steps between 5 and 45 kHz. Previously calibrated values for the probe tube were used to correct the condenser microphone readings to the maximum sound pressure level (in dB re. 20 /xPa) available at each frequency. The corrected values were stored in computer memory and used to calculate the level (in dB SPL) delivered to each earphone during the experiment. Due to the high thresholds of younger animals, voltage levels at the earphones were adjusted to maximal levels for animals less than 14 days of postnatal age, which resulted in a p e a k output of approximately 145 dB SPL between 0.8 and 2.75 kHz (Fig. 1A). The voltage delivered to the earphones was decreased by 20 dB in older animals, in order to decrease the potential for delivery of damaging sounds to the cochlea. Measurements of harmonic distortion made using an acoustic coupler revealed that the first and second harmonics were attenuated by not less than 25 dB and no more than 75 dB relative to the fundamental for all frequencies greater than 100 Hz at the maximum output of the system (Fig. 1B). Intermodulation distortion product levels were at least 45 dB below those of the primaries at all frequencies (Fig. 1C). The noise floor was below 70 dB SPL across the entire frequency range (Fig. 1).

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Recording and data collection techniques

Fig. 1. Acoustic characteristics of the stimulus generation system are shown as a function of fl frequency. The second tone was placed at 1.3 times fl; both signals were presented at 0 dB attenuation. (A). Maximum sound pressure levels. (B). Harmonic distortion levels. (C). Intermodulation distortion levels. The dotted line in each frame represents the noise floor.

Glass micropipettes were positioned visually over the auditory nerve or A V C N with the aid of a surgical microscope. Electrodes were fastened to a hydraulic microdrive (David Kopf Instruments 607W), and advanced from outside the recording chamber following placement. The micropipettes were filled with 2 M potassium acetate (KAc) and typically had tip impedances between 15 and 30 MI2. The recording electrode was coupled to a silver-silver chloride wire and referenced to a ground wire placed in the neck musculature. Recorded voltages were delivered to a preamplifier (Dagan Corporation 8100-1), viewed on a storage oscilloscope (Tektronix D5113) and delivered to an audio monitor (Grass Instrument Company AM8). Event times for individual action potential discharges were digitized in real time using the gate

output of an oscilloscope (Tektronix D10), and transferred to the computer via a second Schmitt trigger (Laboratory Peripheral System). Spike times were measured relative to the onset of the stimulus and stored with 10/xs resolution along with stimulus information in computer files. Neural activity, synchronization pulses, stimulus output and voice commentary were digitized and recorded ( N e u r o D a t a Instruments Corporation DR-484) to allow off-line redigitization of runs which contained noise or other artifacts. Tuning curves were collected using 50 ms tone bursts presented every 100 ms using an automated procedure (modified from Liberman, 1978). This algorithm m a p p e d a frequency span from 45 kHz to 100 Hz in

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J.L. Fitzakerley et al. /Hearing Research 77 (1994) 135-149

steps of 24 points per octave (ppo) for frequencies greater than or equal to 2 kHz and 16 ppo for frequencies below 2 kHz. Acoustic stimuli for the two-tone paradigm consisted of 410 ms tone bursts with 5 ms rise/fall times, presented every 800 ms for a total of 10 to 50 trials (constituting one run). Probe tones ( f l ) were presented at or near the characteristic frequency (CF) of the unit. In general, test tones (f2) were initially presented at 1.3 times CF, then at 0.7 times CF, and then at as many other ratios as time allowed. These parameters have been shown to reliably elicit two-tone suppression in adults (Javel et al., 1978; Schmiedt, 1982; Prijs, 1989). Thresholds in response to the probe and test tone were confirmed using audiovisual methods. The probe tone level was fixed between 0 and 15 dB above audiovisually-determined threshold; in the majority of cases, f l was presented 6 - 1 0 dB above threshold. Test tone levels were generally decremented from 4 dB above f2 threshold (or the maxim u m available stimulus level) to f l threshold, or until no effect of the test tone was observed. In adults, test tone levels were decreased in 4 dB steps; in younger animals, where dynamic ranges were smaller, 2 dB steps were used. In the youngest animals, the ability to produce effective stimulus levels was limited by the maximum output of the stimulus generation system, which complicated the study of two-tone interactions in two ways. First, in the youngest animals, the maximum available acoustic energy limited presentation of the probe tone to a small increment above threshold (i.e. below the 6 - 1 0 dB level typically used in adult animals). This, coupled with the low driven rates of immature neurons in general, made determination of significant changes in response rate problematical. Second, in some cases, there was insufficient acoustic energy to completely characterize the rate/intensity relationship for the test tone, as f2 threshold could not be determined. Most of the two-tone series consisted of a run with the f l tone presented alone, a run with f2 alone, a run with the probe and test tones together (fa +f2), and a repeat of the f l alone condition. The second f l run was used as an index of discharge rate variability, and responses were excluded from the analysis if driven rates in response to the second presentation of the probe tone were significantly different from the first. In some cases, the second probe tone response was not obtained because the unit was lost; these data were included unless there was other evidence that the driven rate of the unit was unstable.

Data analysis procedures Neurons were identified as fibers or primary-like units on the basis of several criteria. Action potential shape (as observed unfiltered during data collection) was used as the initial criterion, with auditory nerve

fiber spikes being identified by their monopolar shape (Tasaki, 1954; Kiang et al., 1965) and primary-like neurons by their prepotentials a n d / o r bipolar characteristics (Pfeiffer, 1966a). In addition, response latencies to CF tones were also employed to differentiate fiber and cell body recordings. Any neuron which produced a bipolar action potential characteristic of an A V C N cell body response was also classified on the basis of its peristimulus time histogram (PSTH) characteristics (Pfeiffer, 1966b; Walsh et al., 1990). Nonprimary-like responses (e.g. chopper) were not included in any of the analyses. The responses of primary-like neurons in the spherical cell region of A V C N have properties very similar to those of auditory nerve fibers (Pfeiffer, 1966b; R h o d e et al., 1983; Rhode and Smith, 1986), and have been used in at least two studies of suppression in adult cats (Rose et al., 1974; Greenwood et al., 1976). A V C N units which showed any evidence of inhibition (such as a decrease in spontaneous rate during presentation of a tone or nonmonotonicity in a rate vs. intensity curve) were also eliminated from subsequent analyses. W h e n data obtained from auditory nerve fibers and from primary-like neurons were plotted independently, no trends were apparent in either data set which were not present in the other. Spontaneous rates were calculated using two methods. In the majority of cases, spontaneous rates were calculated from the last one-third of the off period of the duty cycle (off-period rate) of a run where the stimulus was presented at or below threshold. In later experiments, spontaneous rates were occasionally estimated by collecting spikes over a 10.02 s period with no stimulus presentation. These two methods yielded similar results. Driven discharge rates in response to stimulus presentation were calculated for each run by subtracting the off-period rate from the tone-evoked rate on a trial by trial basis, and averaging this rate across all trials. Trials contaminated by noise or in which spike data were missing were not included in the rate calculations. When comparisons were made among probe, test and two-tone conditions, the mean rates for each condition were computed over the same sequence of trials. Tuning curve analyses were based on the responses of 1448 neurons (1275 fibers and 173 primary-like units). All tuning curve data were smoothed prior to being analyzed. Characteristic frequency was defined for each unit as the frequency having the lowest threshold for producing a detectable increase in discharge rate. Center frequency (computed as the center of the bandwidth of the tuning curve) was used as CF when the tuning curve did not appear to have a definable 'tip' region upon visual inspection. Audiovisually-determined thresholds for the probe and test tones were typically within - 3 _+ 6 dB of the threshold determined

ZL. Fitzakerley et al. / Hearing Research 77 (1994) 135-149

by the tuning curve algorithm (maximum differences - 3 9 and + 24 dB), and corresponded to a driven rate of approximately 5 spks/s in older animals. Tail frequency and threshold were defined in a similar manner to CF and CF threshold, i.e. the tail frequency had the lowest threshold below CF when the tip region was excluded from the analysis. In older animals, when voltages were lowered and an upper limit of 90 dB SPL was placed on the tuning curve algorithm in order to prevent damage to the cochlea, tail thresholds were reported at 90 dB SPL if the tuning curve algorithm failed to determine any thresholds between the end of the tip region and 1 kHz. In all cases, tip-to-tail ratios were calculated as the absolute value of the difference between the tail and CF (tuning curve) thresholds. In the batch analyses, ratios calculated by using the 90 dB SPL limit are termed 'minimum estimated' ratios; ratios based on true tail thresholds are termed 'actual' ratios. Quality (Q) factors were calculated as CF divided by the bandwidth of the tuning curve at a specified level above threshold (i.e. Ql0 calculations were made 10 dB above threshold). Slopes above and below CF were calculated by linear regression on a maximum of three frequency regions both above and below CF, with points of inflection being identified manually. All slopes included in the analyses consisted of at least 3 points, and had correlation coefficient (R) values that were significant at the 0.05 level. The steepest slope above CF (Amax) and below CF (Bmax) were determined for complete tuning curves, and corresponded to the region of the curve closest to the tip in 63% (284/451) of slopes above CF and 68% (290/425) below CF in adult animals. For certain analyses, the responsive frequency range, which spans from approximately 0.1 to 45 kHz in the adult cat, was divided into 5 frequency groups: Group A (apical turn): < 0.851 kHz Group B (middle turn): 0.851-2.750 kHz Group C (lower basal turn): 2.751-7.960 kHz Group D (upper basal turn): 7.961-21.500 kHz Group E (hook): > 21.500 kHz. The boundaries were set at frequencies representing approximately equal basilar membrane distances in the adult, and were determined using the f r e q u e n c y / c o chlear distance relationship described by Greenwood (1961, 1990) and confirmed by Liberman (1982), and the anatomical data of Spoendlin (1972). In some analyses, each of these five groups was subdivided into two smaller frequency ranges in order to increase resolution. The division within each range was again made at approximately equidistant basilar membrane lengths, and occurred at the following frequencies (listed from group A to E): 0.375, 1.58, 4.75, 13.1 and 35 kHz. On the basis of analyses reported in an accompanying paper (Fitzakerley et al., 1994a), two-tone data in this report, but not companion papers, were restricted to

139

those collected from neurons with CFs greater than 0.85 kHz. Two-tone data were obtained from 323 neurons (239 fibers and 84 primary-like neurons), recorded from 59 animals. Data were restricted to cases where f 2 : f l ratios were less than 0.95 or greater than 1.05, and situations where the test tone produced no excitatory response when presented by itself (defined as a driven rate of less than 5 spks/s). Significant changes in rate in response to the two-tone stimulus as compared to the probe tone alone condition were determined by calculating a detectability ratio, d ' = ( D R F I + F 2 DRF1)/(o-21+F2 + ¢r21)°'5, where d' is the normalized rate response (in standard deviations), D R is the driven rate and or2 is the variance in response to the probe or the two-tone condition. Suppression was defined as occurring if the d' value for a given series was less than or equal to + 1, and facilitation if d' was greater than or equal to + 1. This technique for determining significance has its origins in signal detection theory (Green and Swets, 1966), and has been used to evaluate auditory nerve responses to masking conditions (Sinex and Havey, 1986; Mott et al., 1990) and tones in noise (Young and Barta, 1986; Miller et al., 1987), and as an alternative method for threshold determination (Teich and Khanna, 1985; Delgutte, 1988; Kim et al., 1990; Winter and Palmer, 1991). Several techniques have been used to define the common standard deviation in the denominator of the d' calculation. Under ideal circumstances, ~rFl = O'F1+ F2 and both probability distributions are Gaussian. However, as described by several investigators (Teich and Khanna, 1985; Sinex and Havey, 1986; Young and Barta, 1986; Fitzakerley et al., 1991; Fitzakerley, 1992), variance is dependent upon rate, and this assumption, therefore, is not valid. Some authors have used some form of a 'variance stabilizing transformation' to correct for this in conditions where the distributions deviate significantly from ideal conditions (Teich and Khanna, 1985; Young and Barta, 1986). However, it has been argued (Viemeister, 1988; Mott et al., 1990) that such transformations are not necessary for low rates, where the probability of spike occurrences is not being unduly influenced by neuronal absolute and relative refractory periods. This appears to be true for driven rates below approximately 30 spks/s (Geisler et al., 1985; Young and Barta, 1986). In this study, where f l was deliberately placed near threshold, and because rates are generally low in younger animals, the absolute refractory period was not likely to be a limiting factor, and the square root of the sum of the individual variances was chosen as the denominator. The percentages of neurons exhibiting suppression or facilitation were calculated based on whether the criterion d' value was obtained under any of the stimulus conditions being tested in a given analysis. Using

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this criterion, some neurons were tested only once for a given condition, while other neurons were tested several times. This technique may therefore result in an underestimate of the percentage of neurons exhibiting suppression or facilitation in response to a particular condition, as other parameters may not have been optimal for producing significant rate differences.

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3. Results I---

Response categorization Neurons were segregated into three developmental classes, based on responses to single tone stimuli, particularly tuning curve characteristics. Units were considered immature if they failed to meet adult standards established within each CF group for minimal Q5 and Ql0 values, minimal slopes above and below CF, minimal tip-to-tail ratios and maximal CF thresholds (Table 1). A tuning curve was also considered immature if the CF was below 2.970 kHz and it had a tail region. Representative examples of tuning curves obtained from units with CFs in group B (0.851-2.75 kHz) are shown in Fig. 2 in order to demonstrate differences between the three developmental classes, which were: Broadly tuned neurons (type IB). These units had Qs, 010 or steepest slope values which were lower than the adult minimum values. This class of neurons also included untuned units, which responded to long duration stimuli but whose thresholds and CFs could not be determined by the tuning curve algorithm. Sharply tuned, immature neurons (type Is). These neurons exhibited frequency selectivity which met the adult tuning criteria, but possessed other immaturities, such as low tip-to-tail ratios or high thresholds. In this class, the mean and maximum Q and slope values tended to be lower than those of mature units, even though they met the minimum tuning criteria. Mature neurons (type M). These neurons had response characteristics which met all of the criteria outlined in Table 1. However, it should be emphasized that this definition of maturity is based on functional characteristics, not age, and this class therefore includes data collected from animals as young as 75

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Frequency (kHz) Fig. 2. Examples of tuning curves obtained from units with CFs in group B are shown in order to demonstrate differences among the three developmental classes. Of the examples of type I B neurons, the tuning curve with the highest threshold was obtained from a 74 gestational day animal, and the Q values were below the criteria. The example with the lower threshold was obtained from a unit recorded from a 76 day old animal, and the Bmax value failed to meet adult standards. The type I s example was obtained from a 75 day old animal, demonstrated mature frequency selectivity, but had a high threshold and a definable tail region (adult curves in this CF group do not exhibit tails). The example of the type M neuron was obtained from an adult.

gestational days. It was also possible for units recorded during a single experiment to be classified as different developmental types. The percentage of neurons falling into each of the three developmental classes changed with age and with CF group (Fig. 3). Broadly tuned units were most prevalent in the lowest CF groups (A, B and C) at the youngest ages, whereas the majority of immature units in group D (roughly 8-20 kHz) were sharply tuned when first observed. Very few high CF (group E) neurons exhibited any form of immaturity at any age. The time course of maturation was generally similar among the four lowest CF groups, with a relatively rapid decrease in the percentage of type I B neurons, and a more gradual decrease in type I s neurons. However, as a result of differences in the maturational time

Table 1 Criteria used to define mature response characteristics for each CF group Parameter

Qs Q10 Steepest Slope Above CF ( d B / o c t ) Steepest Slope Below CF ( d B / o c t ) Tip-to-tail Ratio (dB) CF Threshold (dB SPL)

CF Group A ( < 0.85 kHz)

B (0.851-2.75 kHz)

C (2.751-7.96 kHz)

D (7.961-21.5 kHz)

E ( > 21.5 kHz)

1.436 0.651 15.0 - 9.6 95

1.555 1.162 33.2 - 15.3 73

3.651 1.940 91.5 - 30.6 9 58

3.693 2.871 64.7 - 46.1 9 71

3.130 2.422 84.8 - 40.1 22 68

J.L. Fitzakerley et al. /Hearing Research 77 (1994) 135-149 • Type Ie

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Gesfational Age (days) Fig. 3. The percentages of acoustically-responsive neurons within each developmental class are plotted as a function of gestational age for each CF group. Only groups where n > 3 are plotted. Data are smoothed. (A). Group A (< 0.85 kHz): n = 3169-72], 16173-75], 22176-78], 13179-81], 11182-84], 5188], 20193-99], 17[103-111], 78[ > 240]. (B). Group B (0.851-2.75 kHz): n = 30169-72], 77173-75], 83176-78],48179-81], 22182-84], 28188], 6193-99], 10[103-111], 88[ > 240]. (C). Group C (2.751-7.96 kHz): n = 14173-75], 54176-78], 49179-81], 8182-84], 14188], 9193-99], 5[103-111], 111119], 1[137], 71[ > 240]. (D). Group D (7.961-21.5 kHz): n = 4173-75], 20176-78], 35179-81], 27[82-84], 1188], 15193-99], 101103-111], 93[119], 215[ > 240]. (E). Group E (> 21.5 kHz): n = 2179-81], 1182-84], 42193-99], 23[103-111], 13[119], 4[137], 57[ > 240].

courses for these neuronal groups, the percentage of type M neurons observed within CF groups A and B ( < 2.751 kHz) gradually increased during the developmental period studied, and the proportional increase of mature neurons with higher CFs (groups C and D) tended to be biphasic. The first sharply tuned units (type I s or M) were observed shortly after birth within all CF groups (i.e., at 70 gestational days for group B, 72 days for group A, 74 days for groups C and D and 81 days for group E). However, the age at which the population in each CF range achieved mature tuning (i.e., was consistently composed of less than 10% type I B units) occurred at 82-84 days for group B, 88 days for group A, 93-99 days for group C, and approximately 119 days for group D. Moreover, the age at which over 90% of the neurons in each CF range displayed complete maturity (based on tuning characteristics) was 93-99 days in

Two-tone interactions Suppression was not observed among the youngest animals studied under stimulus conditions typically producing suppression in adults. However, a small percentage of type I B neurons exhibited facilitation in response to two-tone stimuli. While the characteristics of facilitation are considered in an accompanying paper (Fitzakerley et al., 1994b), an example of such a response, obtained from a broadly tuned auditory nerve fiber, is shown in Fig. 4A. The response to the two-tone stimulus produced an increase in rate of 7.8 s p k s / s (i.e., 1.8 fold greater than the rate to f l alone), with a resultant d' value of 0.99. As can be seen from this example, in cases where neurons exhibited rhythmic firing to the probe tone, which is common among immature auditory neurons, the response to the fa + f2 condition also exhibited a rhythmic pattern. Rhythmicity had no effect on the evaluation of facilitation or suppression, as d' values were based on the separation in the distributions of the probe and combined tone rates calculated over the entire 410 ms presentation interval (as shown in the insets), rather than the fine structure of the rate response exhibited in the peristimulus time histograms. Many immature neurons, whether broadly or sharply tuned, exhibited no significant changes in rate in response to the two tone condition. An example of the absence of significant two-tone interactions in the response recorded from a type I s nerve fiber is shown in Fig. 4B. U n d e r the two tone conditions, the driven rate was reduced by 6 s p k s / s (33%) relative to the probe elicited rate, and the resulting d' value was only - 0 . 8 7 due to the large variance associated with both probe and test conditions. Suppression exhibited by primary-like neurons was indistinguishable from that of auditory nerve fibers, as shown in Figs. 4C and 4D, respectively. In response to two tones, the type M, primary-like neuron shown here exhibited a 29.0 s p k s / s (58%) decrement in rate relative to the control condition, with a d' value of -1.85. For the auditory nerve fiber, which was type I s , the rate produced by the probe tone (i.e., f l alone) was decreased by 32.4 s p k s / s (97%) under two-tone conditions, resulting in a d' value of - 1 . 9 . The parameters which were the most strongly correlated with the presence or absence of suppression were those related to sharpness of tuning. Very few (8%; 3 / 3 9 ) broadly tuned units exhibited suppression in response to test tones placed above CF, and those that did barely met the criterion for suppression (they had d' values of - 1 . 0 to - 1 . 1 ) . These three units were recorded from animals that were 81, 103 and 119 days old, and all had high CFs ( > 14 kHz). In contrast, 31%

J.L. Fitzakerley et aL / Hearing Research 77 (1994) 135-149

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(between 81 and 140 days of age) were similar to those from adults, although mean values were smaller. For a given neuron, the absence of suppression could be predicted on the basis of the sharpness of its tuning curve (Fig. 6). If Q5 values were less than 3, a n d / o r Q10 values were less than 1.5, suppression was not observed. However, above these values, neither suppression magnitude (Fig. 6A,B) or incidence (Fig. 6C,D) was correlated with degree of frequency selectivity. The percentages of units expressing suppression fluctuated around 40% when Q5 values were between 4 and 12, and a lower percentage of units with Q5 values less than 4 or greater than 12 exhibited the phenomenon (Fig. 6C). Although variability was higher when Q10 values were analyzed (Fig. 6D), neurons with Q~0 values less than 1.5 or greater than 9 were less likely to express suppression than units with intermediate levels of frequency selectivity. The majority of units with the highest Q values had CFs greater than 17 kHz, and were recorded from animals older than 93 days of age. The relationships between measures of suppression and tuning were confirmed when tuning curve sharpness was evaluated using the steepest slopes above and below CF. Very few examples of suppression were observed when the steepest slope above CF was less than 100 dB/octave (Fig. 7A) or when the steepest slope below CF was less than 25 dB/octave (Fig. 7B). However, above these values, there was no correlation between tuning curve slopes and either the magnitude of d' or the percentage of neurons exhibiting suppression (Fig. 7C,D). Neurons whose tuning curves had

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J.L. Fitzakerley et aL / Hearing Research 77 (1994) 135-149

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144

J.L. Fitzakerley et al. / Hearing Research 77 (1994) 135-149

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extremely steep slopes below CF ( < - 175 d B / o c t a v e ) showed no evidence of suppression, although the sample size (n = 8) was relatively small. Most of these units had CFs greater than 18 kHz. In units with definable tail regions (which represented a relatively small segment of the total population), there was a tendency for the magnitude of suppression to be related to tip-to-tail ratio (Fig. 8A). Neurons with tip-to-tail ratios less than 10 dB never exhibited suppression, but if ratios were larger than 10 dB, there was no difference in the percentage of neurons exhibiting suppression with increasing tip-to-tail ratio (Fig. 8B). The immaturity associated with low CF units having tail regions did not appear to have any relation to the expression of suppression, as 4 of the 9 units tested which had CFs less than 3 kHz and tip-totail ratios greater than or equal to 10 dB were suppressed by the addition of the second tone. The percentage of neurons exhibiting suppression increased with age within the population of neurons having CFs greater than 0.85 kHz and having probe tone rates greater than or equal to 10 spks/s (Fig. 9). Throughout early postnatal life, neurons exhibiting suppression were mixed with representatives expressing similar CFs that did not respond differentially to two tone conditions, within the same preparation. The first incidence of suppression occurred among relatively low CF fibers (i.e., 2.5-3.7 kHz) at 77 gestational days in response to test tones placed above CF. Although the sample size was small in the youngest age group, the first evidence of suppression in response to a test tone placed below CF was obtained at 80 days. The time course and magnitude of the overall increase in the percentage of neurons exhibiting suppression was similar for test tones placed both above and below CF. In both cases, the percentage of neurons in the tested population which were broadly tuned decreased over approximately the same time course as suppres-

sion appeared. However, there were still differences between the percentage of neurons exhibiting suppression in the population of units recorded in the oldest kitten (137 gestational days, or approximately 2.3 postnatal months) and those of adults, suggesting that the final time course over which suppression develops may be relatively prolonged.

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J.L. Fitzakerley et aL / Hearing Research 77 (1994) 135-149

145

Table 2 Timing of the appearance and maturation of various parameters related to frequency selectivity and suppression for each CF group. N u m b e r s indicate the gestational age (in days), or age groups where the first incidence of each characteristic was observed CF Group

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Developmental changes in the ability to produce suppression, therefore, appear to be linked to the appearance of sharp tuning. However, other factors, such as CF, threshold and spontaneous rate, were also related to the production of suppression, particularly among type I s neurons; these factors are discussed in an accompanying paper (Fitzakerley et al., 1994a). As is detailed in Table 2, within each CF group where suppression was exhibited in adults, the appearance of sharp tuning preceded the appearance of suppression. However, the disappearance of broad tuning (evidenced by a population consisting of less than 10% type I n units), and the final acquisition of adultlike tuning characteristics (defined by having over 90% of the population composed of type M units), occurred much later than either the first observation of suppression or the age that more than 20% of neurons with probe-evoked rates of more than 10 spks/s exhibited suppression.

4. Discussion

On the basis of the results presented here, acoustically-responsive neurons recorded from immature animals were divided into three general classes: 1) immature, broadly tuned neurons (type IB), with low CFs a n d / o r high thresholds, that did not display suppression 2) immature sharply tuned neurons (type Is), with high CF thresholds a n d / o r immature tip-to-tail relationships, that exhibited suppression, but to a lesser degree than mature neurons, and 3) mature neurons (type M), whose tuning and suppression characteristics fell within the range determined for adults. The correlation between suppression and tuning is evidenced by differences in the incidence and magnitude of suppression between broadly (type I B) and

sharply (type I s and M) tuned units. Based on frequency selectivity criteria established from adult data, very few broadly tuned neurons (3/39) exhibited suppression, at any age, and they exhibited rate differences which barely met the d' criterion for suppression. In addition, these units had high CFs, were recorded from animals older than 81 days, and were therefore not representative of the broadly tuned neurons typically recorded from the youngest animals. Excluding those broadly tuned neurons, the first occurrences of suppression were observed at 77 gestational days in two neurons whose tuning met minimum adult standards. The link between tuning and the presence or absence of suppression has also been made in adults, in cases where tuning was altered by damage to the organ of Corti. There is a consensus that complete loss of outer hair cells (OHCs) produces broad tuning and complete elimination of two-tone suppression (Dallos et al., 1980; Schmiedt and Zwislocki, 1980; Schmiedt et al., 1980). However, if the OHC loss is incomplete, the relationship is more complicated, with most studies showing a decreased extent of suppression areas, a n d / o r an increase in suppression threshold in units which were sharply tuned, but whose tuning curves appeared somewhat broader than normal (Dallos et al., 1980; Schmiedt and Zwislocki, 1980; Schmiedt et al., 1980; Mills and Schmiedt, 1983). It has been reported in one study that sharply tuned responses can be obtained from neurons in which two-tone suppression areas were not observed (Robertson and Johnstone, 1981). The results of cochlear lesion studies have also led to the suggestion that suppression areas above CF are related to the high frequency slope of tuning curves, although this hypothesis was formulated on the basis of individual examples (Schmiedt and Zwislocki, 1980; Schmiedt et al., 1980; Mills and Schmiedt, 1983). In the data reported here, there was no direct correlation of

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suppression magnitude or percentage of neurons exhibiting suppression with the steepest slope above or below CF, when a criterion value of 100 dB/octave above CF a n d / o r - 5 0 dB/octave below CF was achieved. Results of the analysis of suppression as a function of Q factor also support the contention that a criterion degree of tuning is required before suppression is exhibited in response to a test tone placed above CF. At all ages, suppression was never observed when Q5 values were below 3 a n d / o r Q10 values were below 1.5, and there was no demonstrable relationship between magnitude or the percentage of neurons exhibiting suppression if Q values exceeded those criteria. It should also be noted that immature, broadly tuned neurons exhibiting suppression had slope and Q values greater than these criterion values, but lower than the minimum values for adultlike 'sharp' tuning for neurons with CFs greater than 14 kHz. One anomalous finding in this study was that some of the most sharply tuned units, those with Q5 values greater than 12, Q10 values greater than 9, a n d / o r maximum slopes below CF of greater than 175 dB/octave, did not exhibit suppression in response to test tones placed above CF. These units, which were obtained from adult animals, were recorded primarily from units with CFs greater than 17 kHz. It has been shown that the sharpness of tuning of suppression areas above CF increases with increasing CF (Sachs and Kiang, 1968; Prijs, 1989), suggesting that the narrow range of test tone frequencies tested on individual high CF units may have contributed to suppression areas being missed in such units in this study. Overall, the percentages of units exhibiting suppression in this investigation were lower than those reported by other investigators. This is an important consideration, given that the lack of suppression observed in young animals represents a negative result. Sachs and Kiang (1968) first demonstrated that suppression areas exist for all auditory nerve fibers, given sufficient recording time to thoroughly test all f2 frequencies and intensities. These results have been confirmed by other investigators (Schmiedt, 1982; Prijs, 1989). This suggests that the restricted test tone ratios employed in this study may have contributed to the lower percentages of suppression observed. Ratios were, however, selected to optimize the detection of suppression in the majority of units, and all neurons were tested initially with test tones with a high probability of producing suppression. It has also been reported that it is more difficult to demonstrate suppression when low probe tone levels, such as those employed in this study, are used (Schmiedt, 1982; Prijs, 1989; Delgutte, 1990a). In particular, smaller suppression areas (Prijs, 1989) and lower rates of suppression growth with increases in test tone intensity (Delgutte, 1990b) have been observed when low probe tone rates

are employed. In this investigation, the high thresholds of younger animals, in combination with the maximum available stimulus levels, limited probe tone levels to within 15 dB of threshold (with the majority being presented at 7-9 dB above fa threshold). These levels, which were used in all animals including adults, are therefore lower than those used in many suppression studies, and may be partially responsible for the lower percentage of neurons exhibiting suppression reported here. Another major difference between the results of these experiments and those of other studies concluding that suppression can be elicited in virtually all adult neurons was the use of a statistical criterion. Using data from test tones placed above CF in this study, 75% of adult neurons show suppression when a criterion of 5 spks/s and 10% difference in rate is used, whereas only 39% exhibit suppression using a d' criterion of - 1 . This d' criterion therefore appears to be a relatively conservative criterion when compared to the rate difference or percentage criteria used in other studies, although this represents a separation of only 68% of the distribution (1 S.D.). A statistical measure was chosen because it is possible that the use of an absolute difference criterion may underestimate the occurrence of suppression for units with low driven rates, such as those recorded from immature animals. A similar problem has been discussed with respect to the estimation of thresholds using rate differences and statistical criteria (Geisler et al., 1985). It should be noted that the presence or absence of a rhythm in the response of immature animals (Walsh and McGee, 1988), does not influence the demonstration of statistical significance, as the variance between trials (not within trials) is used to calculate d'. The contribution of a physiologically vulnerable process which is thought to result in a compressive, basilar membrane nonlinearity appears to be absent, or greatly diminished, during early stages of development, as evidenced by the poor frequency selectivity and lack of suppression which characterizes type I B neurons. In adult animals, two-tone suppression is believed to be mediated by an interference with the active feedback process in the cochlea (Geisler et al., 1990; Cheatham and Dallos, 1992), which is also believed to be responsible for improving frequency selectivity (Neely and Kim, 1983; Geisler, 1991). At all ages and within all CF groups (> 0.850 kHz), the expression of a criterion degree of frequency selectivity was highly correlated with the presence or absence of suppression. This supports the hypothesis that the same mechanism underlies both processes. Additional support is provided by the observation that units with middle to low CFs which exhibited the first evidence of suppression were also the first to demonstrate tuning sharpness within the adult range.

J.L. Fitzakerley et al. / Hearing Research 77 (1994) 135-149

This is a similar interpretation of the developmental process as that proposed on the basis of otoacoustic emission data (Norton et al., 1991). Those authors observed emissions in response to high frequency stimuli prior to those evoked by low frequencies, and suggested that the first significant contribution of the active process is in the cochlear base. The results of single unit studies where adultlike frequency selectivity was observed for fibers with middle to high CFs prior to those with low CFs (Romand, 1983; Dolan et al., 1985; Walsh and McGee, 1990) also support this hypothesis. However, the results of this investigation contrast with these reports as units with relatively low CFs (around 3 kHz) were the first to express suppression and adultlike tuning. There are three potential explanations for these discrepancies. Frequency selectivity (measured in terms of mean and maximum Q and slope values) increases with CF (Evans, 1972; Ohlemiller and Echteler, 1990), even among immature, sharply tuned units (i.e., type I s ) that meet minimum adult standards (Fitzakerley, 1992). If a standard criterion had been used across all frequencies, the results of this study could be interpreted as demonstrating that mature tuning is acquired first for middle to high frequencies. This may be the more relevant measure if suppression, and by implication, the active process, is correlated with a criterion degree of tuning. However, if the minimum degree of frequency selectivity exhibited by neurons recorded from adults is used to define maturity, as it was presented in this study, the first sharply tuned units are observed among low CF groups. Second, if units with low center frequencies which exhibited suppression were, in fact, recorded from more basal locations, as is predicted under the place principle hypothesis (reviewed in Rubel, 1978; Riibsamen, 1992; Walsh and Romand, 1992), maturation of the active process may first be expressed in units with relatively low CFs, but which may reflect different basilar membrane micromechanical processes than those expressed by 'true' low CF units. Third, species differences in the spatial organization of the cochlea, both in the adult and during maturation, may be important. For example, the 2-4 kHz frequency range where the first evidence of tuning and suppression were observed in this study are regarded as low frequencies relative to the adult frequency span in cats (Liberman, 1978), and the definition of 'low' and 'high' CF may be species-dependent. The cochlear process that produces sharp tuning and suppression is thought to involve contraction of the OHCs, which are thought to alter basilar membrane mechanics via interactions with the tectorial and basilar membranes (for review see Dallos, 1988; Yates et al., 1992). There is ample anatomical and physiological evidence that this system undergoes developmental alterations during the acquisition of function. Perhaps

147

the most relevant observation with respect to the current study is that the first primitive connections between the tectorial membrane (TM) and the first row of OHCs in the base of the cochlea are made at birth in the kitten, although the TM does not engage the third row of OHCs until approximately 10 postnatal ( ~ 77 gestational) days (Lira, 1977). The first broadly tuned units, which had CFs between 2.5 and 3.7 kHz, were observed within a few days of birth, and the first suppressive responses were observed approximately 10 days later for sharply tuned units with CFs in that frequency range, indicating that this relationship may be important. The hypothesis that the interaction between the tectorial membrane and OHCs is required for the production of suppression is supported by the observation that two-tone suppression is not produced in the segment of the lizard basilar papilla devoid of a tectorial covering, but does exist for fibers originating in the region where hair cells interact with a tectorial membrane (Weiss et al., 1978; Holton, 1980). However, the presence of type I B neurons (the vast majority of which do not exhibit suppression) and type I s neurons (some of which can be suppressed) with similar CFs in the same animal complicates the identification of a single component of the O H C / T M system that could be responsible for the generation of sharp tuning and the production of suppression. If it is assumed that these neurons were recorded from neighboring cochlear locations, one must look to immaturities in OHC contractile processes, changes in efferent control of the system, or subtle differences in the O H C / T M interactions between near-neighbor inner hair cells to explain such fundamental discrepancies in nonlinear characteristics among units of similar CFs. Although basoapical differences in all of these processes have been described developmentally (for review, see Walsh and Romand, 1992), none have been studied with fine enough resolution to assist in the interpretation of the results discussed here. Several immaturities in the expression of two-tone suppression appeared to evolve over a protracted time course, which was different for each CF group, but ended at approximately 119 gestational (48 postnatal) days. These included differences between type I s and M units in the magnitude of suppression and differences in the ability of test tones presented below CF to produce suppression in neurons within these two classes. These may reflect other immaturities that are considered in an accompanying paper (Fitzakerley et al., 1994a). In addition, the ages at which the population of neurons in each CF group acquired complete maturity (i.e, consisted of more than 90% type M neurons), and at which adultlike percentages of neurons exhibiting suppression were attained, were delayed relative to the time that mature frequency selectivity was demonstrated. This delay may relate more to

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J.L. Fitzakerley et al. / Hearing Research 77 (1994) 135-149

maturational processes associated with the establishment of mature thresholds and discharge rate capabilities than to significant differences in the fundamental mechanisms responsible for producing tuning and suppression (Fitzakerley et al., 1994a). This is supported by the observation that there is an increase in the percentage of neurons exhibiting suppression that is correlated with increases in tip-to-tail ratio. In conclusion, the rate responses of immature peripheral auditory neurons to two-tone stimuli, and the observed improvements in frequency selectivity with age both suggest that signal transduction is linear early in postnatal life in the kitten. The mechanism(s) regl~onsible for producing suppression and sharpness of tuning appears to mature rapidly, with the majority of the changes occurring by 81 gestational (14 postnatal) days. The complexities in the expression of suppression, including their frequency and intensity dependence, appear to reflect interactions between the maturing active process, the changing tonotopic map and the mechanisms involved in threshold maturation in the developing cochlea.

Acknowledgements The authors would like to thank Brian Jacob, Louis Giorgi and Dr. Ye Liu for their technical and surgical support. We also thank Drs. Gorga and Sinex for their critical comments on early versions of the manuscript.

References Cheatham, M.A. and Dallos, P. (1992) Two-tone suppression in inner hair cell responses: Correlates of rate suppression in the auditory nerve. Hear. Res. 60, 1-12. Dallos, P. (1988) Cochlear neurobiology: Some key experiments and concepts of the past two decades. In: G.M. Edelman, W.E. Gall and W.M. Cowan (Eds.), Auditory function: Neurobiological bases of hearing, John Wiley and Sons, New York, pp. 153-188. Dallos, P., Harris, D.M., Relkin, E. and Cheatham, M.A. (1980) Two-tone suppression and intermodulation distortion in the cochlea: Effect of outer hair cell lesions. In: G. van den Brink and F.A. Bilsen (Eds.), Psychophysical, Physiological, and Behavioral Studies in Hearing, Delft Univ. Press, Delft, the Netherlands, pp. 242-249. Delgutte, B. (1988) Physiological mechanisms of masking. In: H. Duifhuis, J.W. Horst and H.P. Wit (Eds.), Basic Issues in Hearing: Proceedings of the 8th International Symposium on Hearing, Academic Press, London, pp. 204-212. Delgutte, B. (1990a) Physiological mechanisms of psychophysical masking: Observations from auditory-nerve fibers. J. Acoust. Soc. Am. 87, 791-809. Delgutte, B. (1990b) Two-tone rate suppression in auditory nerve fibers: Dependence on suppressor frequency and level. Hear. Res. 49, 225-246. Dolan, D.F., Teas, D.C. and Walton, J.P. (1985) Postnatal development of physiological responses in auditory nerve fibers. J. Acoust. Soc. Am. 78, 544-554. Echteler, S.M., Arjmand, E. and Dallos, P. (1989) Developmental

alterations in the frequency map of the mammalian cochlea. Nature 341, 147-149. Evans, E.F. (1972) The frequency response and other properties of single fibres in the guinea-pig cochlear nerve. J. Physiol. (Lond.) 226, 263-287. Fitzakerley, J.L. (1992) Developmental Changes in the Nonlinear Responses of the Peripheral Auditory System, Ph.D. Dissertation, Creighton University. Fitzakerley, J.L., McGee, J. and Walsh, E.J. (1991) Variability in discharge rate of cochlear nucleus neurons during development. Soc. Neurosci. Abstr. 17, 304. Fitzakerley, J.L., McGee, J. and Walsh, E.J. (1994a) Responses of peripheral auditory neurons to two-tone stimuli during development. II. Factors related to Neural Responsiveness. Hear. Res. 77, 135-149. Fitzakerley, J.L., McGee, J. and Walsh, E.J. (1994b) Responses of peripheral auditory neurons to two-tone stimuli during development. III. Rate Facilitation. Hear. Res. 77, 162-167. Geisler, C.D. (1991) A cochlear model using feedback from motile outer hair cells. Hear. Res. 54, 105-117. Geisler, C.D., Deng, L. and Greenberg, S.R. (1985) Thresholds for primary auditory fibers using statistically defined criteria. J. Acoust. Soc. Am. 77, 1102-1109. Geisler, C.D., Yates, G.K., Patuzzi, R.B. and Johnstone, B.M. (1990) Saturation of outer hair cell receptor currents causes two-tone suppression. Hear. Res. 44, 241-256. Green, D.M. and Swets, J.A. (1966) Signal detection theory and psychophysics. Robert E. Krieger Publishing Company, Inc., New York. Greenwood, D.D. (1961) Critical bandwidth and the frequency coordinates of the basilar membrane. J. Acoust. Soc. Am. 33, 13441356. Greenwood, D.D. (1990) A cochlear frequency-position function for several species - - 29 years later. J. Acoust. Soc. Am. 87, 25922605. Greenwood, D.D., Merzenich, M.M. and Roth, G.L. (1976) Some preliminary observations on the interrelations between two-tone suppression and combination-tone driving in the anteroventral cochlear nucleus of the cat. J. Acoust. Soc. Am. 59, 607-633. Guinan, J.J., Jr. and Peake, W.T. (1967) Middle-ear characteristics of anesthetized cats. J. Acoust. Soc. Am. 41, 1237-1261. Holton, T. (1980) Relations between frequency selectivity and twotone suppression in lizard cochlear-nerve fibers. Hear. Res. 2, 21-38. Javel, E., Geisler, C.D. and Ravindran, A. (1978) Two-tone suppression in auditory nerve of the cat: Rate-intensity and temporal analyses. J. Acoust. Soc. Am. 63, 1093-1104. Kiang, N.Y.-S., Watanabe, T., Thomas, E.C. and Clark, L.F. (1965) Discharge Patterns of Single Fibers in the Cat's Auditory Nerve. M.I.T. Press, Cambridge, MA. Kim, D.O., Chang, S.O. and Sirianni, G. (1990) A population study of auditory-nerve fibers in unanesthetized decerebrate cats: Response to pure tones. J. Acoust. Soc. Am. 87, 1648-1655. Kraus, H.J. and Aulbach-Kraus, K. (1981) Morphological changes in the cochlea of the mouse after the onset of hearing. Hear. Res. 4, 89-102. Larsell, O., McCrady, E. and Larsell, J.F. (1944) Development of the organ of Corti in relation to the inception of hearing. Arch. Otolaryngol. 40, 233-248. Liberman, M.C. (1978) Auditory nerve response from cats raised in a low-noise chamber. J. Acoust. Soc. Am. 63, 442-455. Liberman, M.C. (1982) The cochlear frequency map for the cat: Labeling auditory-nerve fibers of known characteristic frequency. J. Acoust. Soc. Am. 72, 1441=1449. Lim, D.J. (1972) Fine morphology of the tectorial membrane: Its relationship to the organ of Corti. Arch. Otolaryngol. 96, 199-215. Lira, D.J. (1977) Fine morphology of the tectorial membrane. In: M.

J.L. Fitzakerley et al. / Hearing Research 77 (1994) 135-149 Portmann and J.-M. Aran (Eds.), Inner Ear Biology, Alnserin, Paris, pp. 47-60. Lippe, W.R. and Rubel, E.W (1985) Ontogeny of tonotopic organization of brain stem auditory nuclei in the chicken: Implications for development of the place principle. J. Comp. Neurol. 237, 273289. Miller, M.I., Barta, P.E. and Sachs, M.B. (1987) Strategies for the representation of a tone in background noise in the temporal aspects of the discharge patterns of auditory-nerve fibers. J. Acoust. Soc. Am. 81, 665-679. Mills, J.H. and Schmiedt, R.A. (1983) Frequency selectivity: physiological and psychophysical tuning curves and suppression. In: J.V. Tobias and E.D. Schubert (Eds.), Hearing Research and Theory, Vol. 2, Academic Press, New York, pp. 233-336. Mott, J.B., McDonald, L.P. and Sinex, D.G. (1990) Neural correlates of psychophysical release from masking. J. Acoust. Soc. Am. 88, 2682-2691. Neely, S.T. and Kim, D.O. (1983) An active cochlear model showing sharp tuning and high sensitivity. Hear. Res. 9, 123-130. Norton, S.J., Bargones, J.Y. and Rubel, E.W (1991) Development of otoacoustic emissions in gerbil: Evidence for micromechanical changes underlying development of the place code. Hear. Res. 51, 73-92. Ohlemiller, K.K. and Echteler, S.M. (1990) Functional correlates of characteristic frequency in single cochlear nerve fibers of the Mongolian gerbil. J. Comp. Physiol. [A] 167, 329-338. Pfeiffer, R.R. (1966a) Anteroventral cochlear nucleus: waveforms of extracellularly recorded spike potentials. Science 154, 667-668. Pfeiffer, R.R. (1966b) Classification of response patterns of spike discharges for units in the cochlear nucleus: Tone burst stimulation. Exp. Brain Res. 1, 220-235. Prijs, V.F. (1989) Lower boundaries of two-tone suppression regions in the guinea pig. Hear. Res. 42, 73-82. Rhode, W.S. and Smith, P.H. (1986) Encoding timing and intensity in the ventral cochlear nucleus of the cat. J. Neurophysiol. 56, 261-286. Rhode, W.S., Oertel, D. and Smith, P.H. (1983) Physiological response properties of cells labeled intracellularly with horseradish peroxidase in cat ventral cochlear nucleus. J. Comp. Neurol. 213, 448-463. Robertson, D. and Johnstone, B.M. (1981) Primary auditory neurons: Nonlinear responses altered without changes in sharp tuning. J. Acoust. Soc. Am. 69, 1096-1098. Romand, R. (1983) Development in the frequency selectivity of auditory nerve fibers in the kitten. Neurosci. Lett. 35, 271-276. Rose, J.E., Kitzes, L.M., Gibson, M.M. and Hind, J.E. (1974) Observations on phase-sensitive neurons of anteroventral cochlear nucleus of the cat: nonlinearity of cochlear output. J. Neurophysiol. 37, 218-253. Rubel, E.W (1978) Ontogeny of structure and function in the vertebrate auditory system. In: M. Jacobson (Ed.), Handbook of Sensory Physiology, Vol. IX. Development of sensory systems., Springer-Verlag, Berlin, pp. 135-237. RiJbsamen, R. (1992) Postnatal development of central auditory frequency maps. J. Comp. Physiol. [A] 170, 129-143. Ruggero, M.A., Robles, L., and Rich, N.C. (1992) Two-tone suppression in the basilar membrane of the cochlea: Mechanical basis of auditory-nerve rate suppression. J. Neurophysiol. 68, 1087-1099.

149

Sachs, M.B. and Kiang, N.Y.-S. (1968) Two-tone inhibition in auditory-nerve fibers. J. Acoust. Soc. Am. 43, 1120-1128. Schmiedt, R.A. (1982) Boundaries of two-tone rate suppression of cochlear-nerve activity. Hear. Res. 7, 335-351. Schmiedt, R.A. and Zwislocki, J.J. (1980) Effects of hair cell lesions on responses of cochlear nerve fibers. II. Single- and two-tone intensity functions in relation to tuning curves. J. Neurophysiol. 43, 1390-1405. Schmiedt, R.A., Zwislocki, J.J. and Hamernik, R.P. (1980) Effects of hair cell lesions on responses of cochlear nerve fibers. I. Lesions, tuning curves, two-tone inhibition, and responses to trapezoidalwave patterns. J. Neurophysiol. 43, 1367-1389. Sinex, D.G. and Havey, D.C. (1986) Neural mechanisms of tone-ontone masking: Patterns of discharge rate and discharge synchrony related to rates of spontaneous discharge in the chinchilla auditory nerve. J. Neurophysiol. 56, 1763-1780. Spoendlin, H. (1972) Innervation densities of the cochlea. Acta Otolaryngol. 73, 235-248. Tasaki, I. (1954) Nerve impulses in individual auditory nerve fibers of guinea pig. J. Neurophysiol. 17, 97-122. Teich, M.C. and Khanna, S.M. (1985) Pulse-number distribution for the neural spike train in the cat's auditory nerve. J. Acoust. Soc. Am. 77, 1110-1128. Viemeister, N.F. (1988) Intensity coding and the dynamic range problem. Hear. Res. 34, 267-274. Walsh, E.J. and McGee, J. (1986) The development of function in the auditory periphery of cats. In: R.A. Altschuler, R.P. Bobbin and D.W. Hoffman (Eds.), Neurobiology of Hearing: The Cochlea, Raven Press, New York, pp. 247-269. Walsh, E.J. and McGee, J. (1988) Rhythmic discharge properties of caudal cochlear nucleus neurons during postnatal development in cats. Hear. Res. 36, 97-116. Walsh, E.J. and Romand, R. (1992) Functional development of the cochlea and the cochlear nerve. In: R. Romand (ed.), Development of Auditory and Vestibular Systems 2, Elsevier Science Publishers, Amsterdam, pp. 161-219. Walsh, E.J. and McGee, J. (1990) Frequency selectivity in the auditory periphery: Similarities between damaged and developing ears. Am. J. Otolaryngol. 11, 23-32. Walsh, E.J., McGee, J. and Fitzakerley, J.L. (1990) GABA actions within the caudal cochlear nuclei of developing kittens. J. Neurophysiol. 64, 961-977. Weiss, T.F., Peake, W.T., Ling, A. and Holton, T. (1978) Which structures determine frequency selectivity and tonotopic organization of vertebrate cochlear nerve fibers? Evidence from the alligator lizard. In: R.F. Naunton and C. Fernandez (Eds.), Evoked Electrical Activity in the Auditory Nervous System, Academic Press, New York, pp. 91-112. Winter, I.M. and Palmer, A.R. (1991) Intensity coding in lowfrequency auditory-nerve fibers of the guinea pig. J. Acoust. Soc. Am. 90, 1958-1967. Yates, G.K., Johnstone, B.M., Patuzzi, R.B. and Robertson, D. (1992) Mechanical preprocessing the mammalian cochlea. Trends Neurosci. 15, 57-61. Young, E.D. and Barta, P.E. (1986) Rate responses of auditory nerve fibers to tones in noise near masked threshold. J. Acoust. Soc. Am. 79, 426-442.