The development of stimulus following in the cochlear nerve and inferior colliculus of the mouse

Developmental Brain Research, 22 (1985) 255-267 Elsevier

255

BRD 50280

The Development of Stimulus Following in the Cochlear Nerve and Inferior Colliculus of the Mouse DAN H. SANES and MARTHA CONSTANTINE-PATON*

Department of Biology, Princeton University, Princeton, NJ08544 (U.S.A.) (Accepted April 2nd, 1985)

Key words: inferior colliculus - - cochlear nerve - - development - - stimulus following - - fatigue - - auditory physiology

The decrement of evoked response amplitudes during the presentation of repetitive clicks was examined quantitatively at the level of the eighth nerve and inferior colliculus in mice aged 13-60 days postnatal. The amplitudes of both these potentials were found to decline during the course of stimulation, this being much more severe at the onset of hearing than in adults. Furthermore the following response at the level of the cochlear nerve was adult-like by day 18, while the response at the level of the inferior colliculus continued to improve through day 24. Recordings in the inferior colliculus were consistently obtained in two different regions along the frequency axis. The regions that responded best to a lower range of frequencies (e.g. 3-9 kHz) showed a more rapid and severe decrement in the evoked response to repetitive stimulation than those regions responding best to a higher range of frequencies (e.g. 8-17 kHz). This was found to be the case for repetitive click stimuli and repetitive tone bursts. Single unit responses in the inferior colliculus were consistent with this differential decline as a function of stimulus rate seen along the frequency axis. INTRODUCTION In the present study, we focus on the d e v e l o p m e n t of temporal p r o p e r t i e s along the m a m m a l i a n auditory pathway. The ability of any sensory neuron to respond to a stimulus is primarily a result of its afferent connectivity p a t t e r n and the synaptic efficacy of those inputs during r e p e a t e d activation. The sensory and neural elements a p p e a r to be especially p r o n e to adaptation or habituation during d e v e l o p m e n t . F o r example, several studies have shown that neurons are able to r e s p o n d to m o r e rapid stimulation rates with increasing age4,5,8,12,19.25,28. The increasing ability of neurons to r e m a i n e n t r a i n e d by repetitive stimuli will have some bearing on the selection of stimulation p a r a m e t e r s in young animals. It will also allow us to better interpret d e v e l o p m e n t a l studies that employ selective stimulation rearing environments. W e have previously used repetitive clicks as a rearing environment in an investigation of the role played by neural activity during the m a t u r a t i o n of frequency tuning26.

One of the specific questions that must be addressed in investigating the d e v e l o p m e n t of hearing is the relative level of m a t u r a t i o n at the p e r i p h e r y and within the central nervous system. M a n y of the connections in the auditory system are p r o b a b l y functional before the cochlea is able to transduce airborne sound36, 38. Consequently, the p e r i p h e r y is likely to represent a m a j o r limiting factor in sensory processing during d e v e l o p m e n t 21. Nevertheless, the maturation of CNS p a r a m e t e r s such as e v o k e d response latency cannot be solely attributed to the chronology of cochlear developmentS, e2. Brugge et al. 3 have r e p o r t e d an e x t e n d e d p e r i o d of m a t u r a t i o n for phase-locking ability in anteroventral cochlear nucleus neurons. This appears to be partly d e p e n d ent upon m a t u r a t i o n within the CNS since the phaselocking ability of eighth nerve fibers is adult-like before the cochlear nucleus attains this state 13. These data suggest that changes in central auditory neurons, such as myelination and synaptic efficacy, could also be involved in sensory maturation. A substantial n u m b e r of d e v e l o p m e n t a l studies

* Present address: Department of Biology, Yale University, New Haven, CT 06511, U.S.A. Correspondence: Dan. H. Sanes. Present address: Department of Otolaryngology, Box 430, University of Virginia Medical Center, Charlottesville, VA 22908, U.S.A. 0165-3806/85/$03.30 (~) 1985 Elsevier Science Publishers B.V. (Biomedical Division)

256 have described stimulus processing in the mouse auditory system 33. Cochlear potentials may first be elicited on postnatal days 8-9 and reach a sensitivity and frequency range resembling that of the adult during days 15-17 (refs. 16, 32). Behaviorally and physiologically measured thresholds do decline slightly beyond this point, however 73:. The N l evoked response latency reaches a minimal value by 20 days of age 32. At the level of the eighth nerve 32, the cochlear nucleus 29, and the inferior colliculus 33, frequency tuning is also well correlated with this rapid developmental sequence. The process of stimulus following and the asymptotic level of maintained response were defined by examining the decrement in evoked response amplitude with increasing click presentation rates. A comparison of the following response at the level of the eighth nerve and the auditory midbrain revealed a differential chronology of functional development. Two issues are examined within the framework of these studies. The first deals with the effect of the duration of stimulus presentation. The second issue concerns the relative ability of auditory neurons to follow stimuli as one moves across the frequency axis of the auditory midbrain. We report here that the evoked response in lower frequency regions of the inferior colliculus decreases more severely and at lower presentation rates than the response in higher frequency regions. MATERIALS AND METHODS

Recordingprocedure Normal C57B1/6J mice at postnatal ages 13, 14, 15, 16, 18, 20, 24, 30 and 60 days were used in the first investigation involving the development of following in the inferior colliculus (IC). Four animals were examined at each age except for the 60-day group (two animals). The mice were first anesthetized with sodium pentabarbitol (Nembutal; 55 mg/kg; i.p.) and placed on a heated stage. The animals head was secured by bonding the skull to a stationary metal plate. Lidocaine was applied to all wounds prior to exposure of the right lobe of the IC. A warm 2% Agar solution was then superfused on the dorsal surface of the IC and the animal was allowed to recover from the surgical level of anesthesia. When the orally monitored temperature began to rise and a small acoustic startle response began to appear, we supplemented with a

dose of chlorprothixene (Taractan; 7 mg/kg, i.p.). During the recording session the animal was maintianed on supplemental doses of pentabarbitol and chlorprothixene. This combination has previously been demonstrated to allow for the recording of hardy response properties in the visual and auditory syste~ns of mice 6,4°. For the N1/N 2 recordings that were obtained from within the cochlear nucleus, we first aspirated the overlying cerebellum. All other procedures were identical to those used in recording from the IC. The entire preparation was housed in a sound attenuation booth (IAC) where acoustic stimuli were delivered closed field (Stax electrostatic earphones). A glass microelectrode (2-3/~m tip i.d.) filled with 2 M NaCl and 1% Fast Green was slowly lowered through the Agar under visual inspection until a click from the audio monitor signified that it first contacted the IC. It was then hydraulically lowered from outside the booth while monitoring the tone-evoked potential. The IC recording sites were in the central nucleus as verified by electrode depth and physiological means. Each site was confirmed to lay in a tonotopic progression of low to high frequency as the electrode moved ventrally. A tonotopic map has previously been generated by comparing single unit characteristic frequencies to electrode depth and Fast Green marks 27. Therefore, only a few electrode sites were marked with Fast Green to confirm our previous observations. A comparison of the present electrode recording depths with those previously obtained for single unit recordings is given below. The signal was amplified and low-pass filtered (WPI DAM-5A), displayed on an oscilloscope, and fed into a signal averager (RC Electronics Computerscope). The averager was triggered at stimulus onset and programmed to accept 20 sweeps for an averaged response. A hard copy of these waveforms was obtained for subsequent amplitude and latency measurements. In order to obtain trigger pulses for counting and poststimulus time analyses, when recording from single units the signal was passed through a window discriminator (WPI 122). Poststimulus time histograms were composed of 100 sweeps.

Stimulus regimen Pure tone pulses were generated by a sine wave os-

257 ciilator (General Radio 1310-A), shaped by an electronic switch, and lasted 15 ms (logarithmic rise/fall time of 5 ms). The click stimulus was generated by a 100/~s rectangular pulse of 1 V. Spectral analysis showed it to be composed of frequencies predominantly below 5 kHz. All stimulus waveforms were passed through an attenuator (Hewlett-Packard 350B) and audio amplifier (Hitachi HA-350) before being fed into the earspeaker. The acoustic output of this system was calibrated with a condenser microphone (Bruel and Kjaer 1/4 in.) coupled to the earspeaker probe tube by a short sealed rubber tube. The RMS voltage and power of the click were computed from the waveform displayed on the oscilloscope and are expressed in dB SPL (0.0002 dyne/cm2). Tonal stimuli were calibrated directly from an RMS meter. All stimuli were presented monaurally, ipsilateral for the cochlear nerve recordings and contralateral for the inferior colliculus recordings. As the electrode was lowered through the IC we monitored the most effective frequency range (i.e. those frequencies that drove a response at lowest threshold) to elicit an evoked potential (EP). We were consistently able to position the electrode such that it sampled predominantly from one of two different frequency ranges: 3 - 9 kHz or 8-17 kHz. The frequency range was determined by observing the EP amplitude to pure tone bursts delivered 20 dB above the dB SPL threshold that had been obtained for click stimuli. In this way, isointensity curves of EP amplitude versus frequency were generated at each recording site. We defined each recording site as being in either a low or a high frequency region. If the EP amplitude was at 50% or less of its peak value (i.e. the largest voltage deviation obtained with the frequency probes) at the frequency ranges 3 and 9 kHz, it was defined as a low frequency recording site. If the EP amplitude was at 50% or less of its peak value at the frequency ranges 8 and 17 kHz, it was defined as a high frequency recording site. Once the frequency range had been determined, we began to test for the following response by presenting a regimen of click stimuli at successively faster rates. The click stimuli were always presented at 20 dB above the click threshold level. This paradigm is illustrated in Fig. lB. Clicks were first presented at 0.1/s and 20 evoked potentials were averaged. After a 2-min silent interval, clicks were presented at a rate

of 0.5/s and an average was obtained. One minute after the onset of this stimulation, 20 evoked responses were again averaged. Stimulation continued and after a total elapsed time of 5 min, 20 final responses were averaged. This procedure was repeated for click repetition rates of 1, 2, 5, 10, 20, 30, 50 and 75/s with 2-min rest intervals between each 5-min presentation (Fig. 1B). In addition, the 0.5/s repetition rate was presented several times during the course of the experiment to ensure that the baseline response had not degraded. It was clear that there was no difference between responses to 0.1 and 0.5/s stimulus presentation rates. Therefore, the averaged response to the 0.5/s click repetition rate taken from stimulus onset was defined as the baseline response. The amplitude of all averaged responses were normalized to the 0.5/s value and, thereby, converted into a percentage (Fig. 1A). In addition, we presented pulsed tones of 5 or 6 kHz and 12 kHz at increasing repetition rates in two animals (20 days). The tone burst stimuli were also presented at 20 dB above their threshold level. The latencies and thresholds of the click evoked responses were also determined for each age group. Latency was defined as the time to the peak of the first large positive-going potential. Threshold was defined as a visually detectable averaged response after 20 click presentations. The stimulus regimen described above was also used to analyze the acoustic following response recorded at the level of the cochlear nucleus. The response consisted of two predominant waveforms 14. The cochlear nerve evoked potential (N1) was difficult to characterize in the frequency domain, as we had done with the EP recorded in the inferior colliculus. It was, however, possible to characterize the frequency response of the second evoked response, the N 2 potential, prior to the presentation of click stimuli. The N 2 potential is probably of both cochlear nerve and cochlear nucleus origin14. 20. The electrode was always positioned such that the frequency range of-the N 2 potential was maximal between 8 and 17 kHz. We also quantified the following response of 29 single units in the IC of 3 animals, aged 19-21 days, to help in the interpretation of the averaged EPs. A repetition rate of 20 clicks/s was presented for 1 min and the evoked spikes were counted at the beginning

258 and the end of the regimen. Again, the clicks were presented at 20 dB above threshold. In a few units we compared the 20/s spike counts to those obtained with a repetition rate of 0.5/s. The only units that were analyzed were those that continuously responded to a click repetition rate of 2/s.

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A n evoked potential (EP) is a relatively slow change in voltage (e.g. compared to an action potential) that is presumed to reflect the synchronous discharge by many neuronal elements. Unlike single units, which respond in an all-or-none fashion, the EP often varies in amplitude in proportion to stimulus intensity. It is presumed that EP amplitude increases as individual units with different thresholds are recruited by the stimulus, although synaptic contributions may add to the response. The ability of neurons in the IC to respond to repetitively presented stimuli was inferred from the relative amplitude of the click evoked response (Fig. 1). A decrease in amplitude was taken to indicate that some neurons were no longer responding to the stimuli. The youngest age at which we were reliably able to quantify this

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Fig. 1. A schematic of the experimental paradigm. A: the first 20 compound action potentials, evoked by clicks presented at 0.5/s, were averaged and the resultant amplitude was defined as the maximal response. For increasing click repetition rates the amplitude of an averaged response was compared to the maximal response and expressed as a percentage. In the example shown, Y is the original averaged amplitude and X is the averaged amplitude to a presentation rate of 30/s. B: the stimulus regimen for each electrode recording site in an animal consisted of increasing click presentation rates as illustrated. Except for the first presentation rate of 0.l/s, all repetition rates lasted for 5 min with a 2-min rest period between each one. Twenty click-evoked potentials were averaged at presentation onset, after 1 min, and after 5 min as indicated by the arrows.

AGE

116

118

(postnatal

20

214

310

610

day)

Fig. 2. Click-evoked potential amplitudes. The amplitudes of evoked potentials recorded in both the low and high frequency regions of the IC are shown plotted against increasing age of recording. Means and standard deviations are plotted. process of response decrement was 13 days postnatal.

Evoked potential shape, magnitude, and recording depth The characteristic waveshape of a click evoked potential in the mouse IC is shown in Fig. 1A. It consisted of a small initial biphasic fluctuation followed by a much larger positive voltage deviation. The large positive potential was used for analysis and probably consisted largely of postsynaptic discharges. We note that unit discharges were often seen 'riding' on the peak of this potential, and its latency was very similar to the single unit latencies that we have obtained previously zT. The magnitude of the EPs varied across age. This is shown in Fig. 2. For 13- to 14-day animals it was approximately 125 ~V. By day 20 this value had risen to approximately 300/~V. The magnitude seemed to decrease slightly after this point, but the results are rather ambiguous for 60-day animals. There did not appear to be any consistent difference in the EP amplitude as recorded in either the higher or lower frequency regions of the IC. The mean recording site depth of lower frequency regions was 454 ~m (S.D. = 106) for animals 20 days or older. By comparison, the mean depths for single units with characteristic frequencies of 4, 5, 6 and 7 kHz were between 400 and 498/~m 27. The mean recording site depth for higher frequency regions was 940 ktm (S.D. = 165). For comparison, the mean depths for single units with characteristic frequencies of 10, 11, 12, 13, 14, 15 and 16 kHz were between 665 and 9 3 4 # m e7.

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Fig. 3. Three-dimensional plots illustrating the following response as a function of click repetition rate and age. A-C: the 3 graphs show percent maximal response for electrode recording sites that were characterized as responding to frequencies in the 8-17 kHz range (HI). D-F: these 3 graphs show percent maximal response for electrode recording sites that were characterized as responding to frequencies in the 3-9 kHz range (LO). The averaged responses were obtained either from stimulus onset (A, D), after 1 rain of stimulation (B, E), or after 5 min of stimulation (C, F). All graphs show a general reduction of response amplitude with increasing click repetition rate or decreasing age. It is clear that LO recording sites fatigued at lower presentation rates than HI recording sites.

260 TABLE

I

Percent maximal response of the inferior colliculus compound action potential to increusing click repetition rates

Age (days)

Time (min)

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15

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16

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18

0

107.0

20

0

100.7

24

1 5 0

97.7

30

5 0

96.6

60

0

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100.0 96.5 98.0 100.0 102.0 94.2 100.2 100.2 97.2 100.0 99.0 100.5 100.0 100.5 101.5 100.0 101.0 102.7 100.0 96.2 95.2 100.0 100.7 100.0 100.0 100.5 98.0

97.5 89.7 89.0 93.2 97.5 91.5 94.2 90.0 90.5 98.7 97.7 95.2 99.7 102.7 104.0 99.5 99.5 100.2 99.0 100.0 101.0 102.5 102.0 102.2 103.5 101.0 101 .o

85.2 79.7 79.0 84.3 77.6 72.3 94.2 89.7 87.5 93.7 89.0 87.7 99.2 97.0 101.2 98.5 93.5 95.5 98.7 93.5 93.2 102.5 101.2 99.0 106.0 102.0 105.0

82.0 66.2 67.0 77.5 70.2 67.5 83.7 78.7 80.0 87.5 85.2 74.0 101.7 92.5 91.7 99.0 95.5 91.5 94.5 88.0 92.2 94.0 93.5 96.5 98.0 98.0 94.5

61.5 42.5 43.2 58.0 45.5 44.7 71.2 58.2 60.2 66.0 61.5 63.2 91.7 77.5 77.5 89.2 84.0 84.5 82.7 77.7 78.7 83.5 78.5 79.0 97.0 88.5 84.5

50.2 32.2 31.7 55.0 34.0 28.5 67.5 53.7 50.7 67.5 54.2 47.7 85.5 67.0 67.0 87.2 77.7 72.2 84.2 79.2 75.7 85.0 81.7 80.0 91.0 83.5 83.5

35.5 23.2 19.6 39.5 25.5 32.0 56.7 39.7 38.5 60.0 13.7 38.0 69.2 55 ._7 __ 57.0 67.5 60.7 61.5 75.7 73.5 69.5 77.2 77.1 78.7 82.5 79.5 73.0

37.7 15.7 10.0 32.7 18.3 15.0 61.2 35.0 39.2 64.5 37.5 38.5 71.7 49.0 51.7 75.2 63.5 66.7 87.7 86.5 88.5 91.7 97.7 95.7 95.5 93.5 93.0

0.0 0.0 0.0 37.0 13.0 10.0 -10.0 28.7 61.0 38.3 23.0 28.5 62.0 43.7 41.5 62.7 56.7 63.3 73.u 78.0 88.2 86.0 95.2 98.7 85.0 93.5 94.5

90.0 77.3 80.6 91.3 86.3 78.3 94.0 91.6 89.0 95.3 95.0 96.0 93.3 95.3 90.6 95.6 98.3 98.3 98.0 93.3 98.0 93.0 93.0 93.0

83.0 77.5 66.5 87.3 74.3 74.3 84.0 79.6 66.3 94.6 81.6 86.3 90.6 83.3 79.3 98.3 88.0 87.6 92.3 86.6 89.6 87.0 90.0 91.5

65.3 52.0 49.0 69.0 54.6 49.3 67.0 60.0 59.0 86.6 79.6 71.3 76.3 70.0 66.0 85.6 81.3 78.3 80.6 77.0 81.0 85.5 82.5 81.5

49.6 25.6 24.3 48.0 29.6 26.6 57.6 38.0 34.6 69.3 51.0 51.3 62.0 44.3 43.6 71.3 56.6 60.0 70.3 56.0 59.6 78.5 63.0 61.5

38.0 15.5 10.0 35.6 10.3 10.0 42.0 21.6 21.0 64.6 38.3 35.3 49.0 26.6 29.0 60.6 38.6 38.6 76.6 55.6 52.0 62.0 52.0 45.5

22.5 10.5 10.0 20.6 5.5 5.0 33.3 17.0 18.0 48.0 27.3 30.5 35.3 20.6 25.0 51.3 31.3 29.3 64.0 43.0 38.3 53.0 34.0 37.0

10.0 5.0 5.0 0.0 5.0 0.0 14.6 1.3 1.0 30.0 24.5 23.5 29.0 14.0 21.0 37.3 21.3 21.3 57.0 44.3 40.3 46.5 28.0 28.0

5.0 5.0 5.0 0.0 0.0 0.0 5.0 0.0 0.0 25.5 15.0 6.0 12.0 3.5 2.0 24.0 15.6 13.0 48.0 35.3 39.6 28.0 27.5 26.0

Lower frequency recording site: 3-9 kHz 14

0

105.5

15

5 0

97.5

16

0

107.0

18

0

95.3

20

5 0

99.6

24

5 0

95.0

30

0

97.6

60

5 0

97.5

100.0 93.0 93.0 100.0 93.0 90.0 100.0 103.6 93.0 100.0 94.0 100.0 100.0 89.3 95.3 100.0 97.3 99.6 100.0 86.3 92.0 100.0 95.5 97.5

261

Development of the following response in the higher frequency region of the inferior colliculus The development of stimulus following was first characterized in the IC for neurons responding best to frequencies from 8 to 17 kHz (see Materials and Methods). The results of these analyses are presented graphically in Fig. 3 A - C , while Table I contains the actual measurements. For click repetition rates of 0.1, 0.5 and l/s, there was very little response decrement at any age studied, either initially or after 5 min of stimulation. With a repetition rate of only 2/s, however, a response decrement of approximately 20% occurred in 13- and 14-day animals. At increasing presentation rates the 13- and 14-day animals exhibited an especially severe decrease in response such that at a rate of 75/s, the averaged EP was negligible. By day 18, however, only repetition rates of 50 and 75/s led to response decrements that were greater than 45% below adult values. The percent maximal response was within 10% of adult values for all presentation rates at postnatal day 24 (Table I). The response decrement after 5 min of stimulation was, with rare exception, 0 - 2 0 % below the initial (e.g. time = 0) value for a given presentation rate. It is clear from the similarity between graphs plotting response amplitude at 1 min and 5 min (Fig. 3B and C) that response decrement had reached an asymptotic minimum by 1 min of stimulation. Animals 24 days of age and older showed a response decrement at relatively low repetition rates, 10, 20 and 30/s, followed by a response increase as the stimulation rate was raised from 30 to 75/s (Figs. 3C and 4C). This non-monotonic response to click rate was not seen in young animals; nor was it apparent for the population of IC neurons characterized as responding to a lower range of frequencies (see be-

low). Development of the following response in the cochlear nerve Having generated a description of stimulus following at the level of the auditory midbrain during development, it was of interest to compare this to the maturation of the primary afferent population. The amount of response decrement exhibited by the N 1 component of the evoked potential recorded in the cochlear nucleus was examined in animals at 14, 18 and 30 days postnatal. In addition, the decrement

of the N 2 potential was also quantified. A comparison of the N1 and IC following responses is shown in Fig. 4. The ability of the auditory nerve to follow click stimuli was relatively mature for repetition rates of up to 10/s at the youngest age examined, 14 days (Fig. 4A). By day 18, the percent maximal amplitude of N 1 after 5 min of stimulation was within 5% of the values obtained at day 30. An exception was the response at 30/s, which we do not entirely understand (Fig. 4A). The N 1 following response, therefore, appears to mature most rapidly between the ages of 14 and 18 days postnatal. We include data obtained from the N 2 potential (Fig. 4B) because we were able to characterize this potential as having been evoked by a discrete range of frequencies (i.e. 8-17 kHz). By comparison, the following response at the level of the IC has a somewhat different ontogeny. At 14 days of age the degree of response decrement is much greater than that seen at 30 days for all click repetition rates above 1/s. This is shown in Fig. 4C. While the response in the nerve has approximated the mature state by day 18, the IC is only able to follow click repetition rates up to 10/s at the adult level (Fig. 4C). Therefore, the ability of IC neurons to follow click repetition rates of 20/s and above develops to a mature level over an extended period compared to the N 1 potential.

Development of the following response in the lower frequency region of the inferior colliculus A second population of neurons in the IC, those characterized as responding to the range of frequencies 3 - 9 kHz, were analyzed with the same stimulus regimen (Fig. 1B) except that animals 13 days postnatal were not examined. The most dramatic finding was that the averaged EP exhibited a decrease in amplitude to lower stimulus presentation rates. This decrease was more severe than for averaged EPs from the higher frequency regions of the IC. This can be seen by comparing the graphs in Fig. 3B and E. The following response showed little, if any, decrement in amplitude for click repetition rates of 0.1 or 0.5/s (Table I). For presentation rates of 5/s and above, the averaged EP amplitude declined approximately 20-40% below the level observed for the higher frequency regions. The decline in response amplitude over 5 min of stimulation was 0 - 2 0 % below the ini-

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Fig. 4. A comparison of the following response at the level of the cochlear nerve and the IC at 3 postnatal ages: 14, 18 and 30 days. The graphs plot percent maximal response after 5 rain of stimulation. A: response decrement of the Ni potential. B: response decrement of the N2 potential. C: response decrement of the EP recorded in the inferior colliculus. The means and 95% confidence intervals are illustrated. It is clear that the N1potential has an adult-like response by day 18, while the IC response is not yet adult-like for the higher repetition rates at this age.

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tial (e.g. time = 0) value, for a given presentation rate. Two additional experiments were performed in which the probe stimuli were chosen for a more selective activation of IC neurons• These were pure tone bursts of low (5 or 6 kHz) or high (12 kHz) frequency, The 5 or 6 kHz stimuli were used for electrode recording sites characterized as being in a lower frequency region, and the 12 kHz tones were used in higher frequency recording sites. Fig. 5 illustrates the results of the two experiments and clearly shows that the averaged potential evoked by the 5 or 6 kHz stimuli declined in amplitude far more than the averaged potential evoked by the 12 kHz probe at the intervals

Rate

(pulses/sec

I

Fig. 5. A comparison of the following response to repetitive tone bursts of 5-6 kHz or 12 kHz. The top row of graphs plot data from two electrode recording sites, characterized as responding to a low or high frequency range, in a single animal. In this experiment a probe tone of 6 kHz was repetitively presented at increasing rates when the electrode was in the low frequency region (i.e. 3-9 kHz). The averaged response from 20 evoked potentials for a repetition rate of 0.5/s at stimulus onset was defined as the maxima] response amplitude and all

other averaged responses were expressed as a percentage of this value. The stimulus regimen was repeated with a 12 kHz tone when the electrode was in the high frequency region (i.e. 8-17 kHz). The bottom row of graphs plot the data obtained from another animal, except that the lower frequency probe tone was 5 kHz, instead of 6 kHz. For both experiments, an averaged response was obtained at stimulus onset, after 1 min of stimulation, and after 5 rain of stimulation. It is clear that the evoked response to lower frequency tonal probes decreases more rapidly than the response to higher frequency probes.

tested. At a presentation rate of 50/s the averaged EP was very small for both frequency probes, most likely reflecting the smaller interval between the tone burst stimuli, as compared to the click stimuli of the same rate.

Stimulus following by single units in the inferior colliculus To provide support for the interpretation that the decrement of the averaged E P in the inferior colliculus actually reflects a response decrement of individual auditory units, we recorded from single units in the IC. Their response decrement over the course of 1 min of stimulation was examined. Fig. 6 shows poststimulus time histograms from two units in the lower frequency region of the IC and two units in a higher

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er than 60% of the original response (e.g. from stimulus onset) to 20 clicks/s. Of the units with higher characteristic frequencies, 14 of 16 showed greater than 60% of the original response. The parameters of characteristic frequency and percent of response remaining were found to co-vary (linear regression; r = 0.47).

Evoked potential latency and threshold The thresholds and latencies of EPs were also obtained to click stimuli in both the low and high frequency regions of the IC. It is apparent from Fig. 8A that thresholds declined rapidly between 13 and 16

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Fig. 8. The development of auditory thresholds and latencies to click stimuli in the IC. A: the decline in mean threshold with increasing age is plotted for both low and high frequency regions of the IC. Threshold was defined as the visual detection level upon averaging 20 click-evoked potentials. An adult value is obtained by about 16 days. B: the decline in mean latency with increasing age is plotted for both low and high frequency regions of the IC. Values decrease rapidly until about day 20 and attain adult values between days 24 and 30. Mean and 95% confidence intervals are plotted. days, irrespective of frequency range. They then appeared to remain relatively constant over the next 40 days. In contrast to this developmental chronology, the latencies of the click-evoked potentials started to decrease on days 15-16 and reached adult levels by days 24-30 (Fig. 8B). The greatest decline in latency, however, occurred from 13 to 20 days of age. Thus, at postnatal day 18, thresholds are adult-like, while latencies are 2-3 ms longer than adult values. DISCUSSION

The present results describe a chronological development of stimulus following that roughly parallels other aspects of maturation in the mouse auditory system32,34. The decline in the amplitude of the repetitively evoked potential indicates that, of those neu-

rons responding to the clicks, the ability to follow stimuli matures from hearing onset until at least 24-30 days postnatal (Fig. 3; Table I). This is true for both the lower and higher frequency recording sites in the IC. While the threshold to click stimuli decreases to adult levels by days 16-18 (Fig. 8A), the decline in response latency (Fig. 8B) has a slower developmental time course, resembling that of the following response. Disparities in the maturation rate of several functional parameters reflect different rates of cochlear and CNS development. The maturation of response threshold occurs very rapidly in all mouse auditory regions examined and appears to be fully contingent upon external, middle, and inner ear maturation. Depending upon the means of assay, response thresholds are within a few decibels of the adult state by days 15-18 (refs. 7, 16, 29, 32). In contrast, the response latency of central neurons seems to be only partially attributable to cochlear development. Shnerson and Pujo132 have reported that toneevoked N] potentials attain their minimal latency by day 20 or earlier, while Mikaelian and Ruben16 have reported a minimal latency to click stimuli by day 14 in a different strain of mice. The present findings on the developing EP in the inferior colliculus show that the latency of clickevoked potentials continues to decrease through days 24-30 (Fig. 8A). Shah et al. 31 have described a similar developmental chronology in the rat. While these authors have implicated myelination as the causative factor, other parameters, such as axon caliber and synaptic efficacy, also mature during this time. It is potentially significant that the development of the Nj and cortical potential latencies in kitten also show different developmental chronologies of this typeS.22. Frequency selectivity, as analyzed with compound action potential tuning curves, may also differ in developmental rate at the level of the eighth nerve and cochlear nucleus 29,32, but dissimilarities in stimulus presentation prevent any firm conclusions from being drawn. It has also been shown in kittens that phase-locking of neurons to low-frequency stimuli develops later for anteroventral cochlear nucleus neurons than at the level of the eighth nerve 13. Although it seems reasonable to expect differences in fatigue between the primary afferent pop-

265 ulation and a central population several synapses away n, it was not clear whether there would be a difference in the rate of maturation. Experiments on the development of following at the level of the cochlear nerve and nucleus (i.e. N 1 and N 2 potentials), when compared to those in the IC, demonstrated that maturational differences do exist as one ascends the mouse auditory system. One indication of this was obtained directly after the onset of hearing. At 14 days the N 1 response exhibited much less response decrement than the click-evoked potential in the IC at all presentation rates above 1/s (compare Fig. 4A and C). Another sign of maturational incongruity is that the N 1 following response has closely approximated the adult state by day 18 for all presentation rates tested. However, stimulus following in the IC remains attenuated for all presentation rates above 20/s in 18-day animals (compare Fig. 4A and C). Although the N 2 potential was qualitatively similar tO N 1 in response to clicks, it was generally of larger relative amplitude in 14- and 18-day animals. The possibility exists that eighth nerve units respond only at longer latencies as the stimulus regimen continues. This implies that the N 2 potential may not decrease in amplitude, as does N 1, because additional units fire at that time. In the IC of older animals, aged 24 days and above, the EP was observed to decrease and then increase in amplitude as click repetition rate increased (Figs. 3 A - C , 4C, Table I). It is not yet known whether the response increment in the mouse IC is due to an artifactual summation of waveforms or to a real increase in responsivity at certain click repetition rates. A response increment at certain stimulus presentation rates has been reported to occur in the cochlear nucleus and medial geniculate nucleuslS.23. 24. A summation of waveforms is suggested from human EPs. As stimulus repetition rate increases from 1-40/s, the middle latency response first decreases in amplitude and then summates, leading to a supramaximal Ep9. This 40 Hz event-related potential is due to the fact that a stimulus-evoked scalp potential consists of several cycles of a 40 Hz sine-wave. The EPs recorded in the inferior coUiculus of mice had a latency of 9-11 ms in older animals, and would not be expected to overlap at the stimulus rates we employed. Furthermore, it is apparent that response decrement followed by response increment is not present in mice 20

days and younger (Fig. 3 A - C ) , nor is it present in regions of the IC responding to the 3 - 9 kHz frequency range (Fig. 3 D - F ) . Our single unit recordings suggest 3 reasons for the decrease in EP amplitude during repetitive stimulation. It is possible that units stop firing completely, that they respond to fewer of the stimuli, or that they fire fewer spikes to each stimulus. It is also possible that firing could become less synchronous, thus leading to a decrease in EP amplitude. Willott and Urban 4o have reported that 47% of the units in the central nucleus of the IC respond to clicks, and we have observed units that will respond to only the first few clicks in a stimulus train, although these were not analyzed in the present study. Of the units that did not habituate immediately, almost all showed a decrease in the number of stimuli to which they would respond and/or fewer spikes to each click (Figs. 6 and 7). A most unexpected finding was that the IC did not show uniformity in its ability to follow repetitive stimuli: low frequency regions showed a more severe response decrement and did so at lower presentation rates. It was generally observed that for presentation rates above 5/s, the amplitude of lower frequency averaged EPs decreased at least 20% more than those in regions characterized as responding to higher frequencies. This was true regardless of age examined when one observed the response after 1-5 min of stimulation (Fig. 3; Table I). For the greater presentation rates, 50 and 75/s, the difference between lower and higher frequency regions in mature animals was 50-70%. One possible explanation for this result is that the EP was composed of units not only from the lower frequency laminae of the central nucleus of IC, but also of units from the pericentral or external nuclei of the IC. These latter divisions are known to contain broadly tuned units that habituate rapidly to repetitive stimuli1.34.39, and these may have accounted for the rapid decline in amplitude. However, two lines of evidence argue against this explanation. First, pure tone burst stimuli, which drive a more selective population of units, were found to give the same difference across the frequency axis. That is, the EP to 5 or 6 kHz repetitive tone bursts habituated more rapidly than the EP to the 12 kHz tone bursts (Fig. 5). Secondly, single unit recordings indicated that neurons

266 with characteristic frequencies below 10 kHz habituated more severely to repetitive clicks of 20/s than those with higher characteristic frequencies (Figs. 6 and 7). These units had narrow frequency tuning curves as are c o m m o n for neurons in the central nucleus of the IC. W e believe, therefore, that the difference in response d e c r e m e n t occurs across the frequency axis of a fairly h o m o g e n e o u s subnucleus of the IC, the central nucleus. We have d o c u m e n t e d a difference in the ability of low and high frequency regions of the IC to follow repetitive stimuli. In this respect, it is interesting to note that mice p r o b a b l y process interaural intensity differences ( I I D ) best for frequencies above 10 k H z 30. It is also known from behavioral studies in rats that a frequency of 8 k H z is much less localizable than higher or lower frequencies ~5. M o r e recently, an anatomical study in the cat described a differential projection from the lateral superior olivary nucleus (LSO) to the IC 10. The lower frequency regions of the L S O project primarily to the ipsilateral central nucleus of the IC, while the higher frequency limb of LSO projects contralaterally. G l e n n d e n n i n g and Masterton 10 suggest that the higher frequency interaural intensity cues essential for sound localization 17, that are initially processed by L S O 2.37, are p r e d o m i nantly parcelled to the contralateral side. The correlation between a f r e q u e n c y - d e p e n d e n t acoustic

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chiasm, the present physiological data on frequency differences in the IC, and the frequency range most likely used by rodents to localize sounds with I I D s , suggest a functional division along the frequency axis of the central nucleus of the IC based on the two most useful binaural localization cues: interaural time and intensity differences. A t present, however, too little information exists to make this more than interesting speculation. In summary, we have quantified the d e v e l o p m e n t of stimulus following at two levels of the auditory pathway. These results indicate that auditory processing matures m o r e rapidly at the periphery than in the central nervous system. W e have also shown that the inferior colliculus does not a p p e a r to be homogeneous in its ability to respond to repetitive stimuli, and that this difference is manifest across the frequency axis. ACKNOWLEDGEMENTS This work was s u p p o r t e d by N I H Training G r a n t 5T32 GM07312 and Sigma Xi G r a n t - i n - A i d ( D . H . S . ) and the Deafness Research F o u n d a t i o n (M.C.-P. and D . H . S . ) . W e thank Dr. E. Rubel for his advice and support and Drs. J. Paton, E. R u b e l and L. Kitzes for critical reading of the manuscript.

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