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1Department of Communication Sciences and Disorders, School of Allied Health Science, East Carolina University, Greenville, North Carolina; and 2Department of Veterinary Biomedical Sciences, School of Medicine, University of Missouri–Columbia, Columbia, Missouri
Submitted 9 June 2005; accepted in final form 3 April 2006
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ABSTRACT |
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INTRODUCTION |
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; Rubel and Fritzsch 2002; Rübsamen and Lippe 1998; Sanes and Walsh 1998) that hearing begins very early in the chicken, commencing on or about day 12 of incubation (E12) shortly after the formation of afferent synapses (Cohen and Fermin 1978; Hirokawa 1978; Rebillard and Pujol 1983; Whitehead and Morest 1985a,b). The pioneering physiological work of Saunders and colleagues (1973) is generally cited as the basis for this notion. Moreover the case has been made that central processing of auditory information also begins at this time (Jackson et al. 1982; Pettigrew et al. 1988; for review see Sanes and Walsh 1998). These fundamental concepts continue to shape the thinking of investigators and influence interpretation of developmental events in central auditory neural pathways.
Despite the broad acceptance of these ideas, direct study of cochlear ganglion neurons in young embryos does not support the hypothesis that "hearing" begins as early as E12 (Jones et al. 2001). At ages younger than E16 (stages 38–42; Hamburger and Hamilton 1951) the intensity levels of natural ambient sounds are generally well below the threshold levels for primary afferents. Therefore we regard this early period as being "prehearing." The question remains then, when are cochlear primary afferents capable of encoding natural ambient sounds in the chicken? This question was among those addressed in the current study.
Rubel and Rebillard (1981) provided evidence that the collective response of the auditory nerve in the E17–E19 embryo exhibited frequency tuning characteristics. Studies of the tuning characteristics of individual ganglion neurons in the late chicken embryo (E18 and E19, stages 43 and 44) also demonstrated that most cells produced frequency tuning curves (FTCs) comparable to those found in mature animals (Jones and Jones 1995a,b). It follows therefore that the emergence of frequency selectivity must begin prior to stages 43 to 44. Determining the approximate onset age for frequency selectivity was a second aim of the present work.
The highest characteristic frequencies (CFs) reported for primary afferent as well as central relay neurons in the adult and hatchling chicken range from about 2,600 to 5,000 Hz (Chen et al. 1996; Coles and Aitkin 1979; Jones and Jones 2000; P6–P14: 4,545 Hz: Manley et al. 1991; P1: 2,640 Hz, P21: 3,303 Hz: Plontke et al. 1999; Saunders et al. 1996; P0: 3,500–3,700 Hz: Warchol and Dallos 1990). Studies in embryos to date have reported the absence of primary afferent CFs 
2,200 Hz (primary afferents: Jones and Jones 1995a,b; central relays: Lippe and Rubel 1983, 1985). The absence of primary afferent neurons with high-frequency CFs in late embryos has been hypothetically attributed to a functionally immature base, reduced transfer of high-frequency sound to the cochlea, and/or technical difficulties that prevent the assessment of the cochlear base (e.g., Jones and Jones 1995b; Manley 1990; Manley et al. 1985, 1991; Salvi et al. 1992). The research reported here was undertaken to critically examine these hypotheses.
In the present study we explored the time course for the emergence of primary afferent responses to sound over the ages of E12 to E18. To examine the role of the middle ear during ontogeny, we measured primary afferent responses produced by normal airborne sounds and compared them to responses obtained with acoustic vibrations applied directly to the columella footplate. Thus we contrasted cochlear function in the presence and the absence of the middle ear.
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METHODS |
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,b, 2000; Jones et al. 2001). Embryonic ages (equivalent days of incubation, E-days) and staging followed the conventions outlined by Hamburger and Hamilton (1951). The "+" symbol given in association with a stage number such as "41+," denotes "late stage 41" and this was represented quantitatively as a half stage (e.g., 41.5). A list of embryonic stages and equivalent "days of incubation" are summarized in Table 1. Chickens (Gallus domesticus) were studied as embryos at equivalent incubation days from E12 to E18 (embryonic stages 39 to 44) and as hatchlings from posthatch days 13 to 15 (P13–P15). In no case did embryos display efforts of pulmonary ventilation. Unless otherwise stated values herein are presented as means ± SD (n = sample size). A significance level of P < 0.05 was used for hypothesis testing.
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Eggs were placed on a heated platform in a sound-attenuating booth. To anesthetize embryos, EquiThesin was diluted 1:5 with normal saline and 0.1 ml administered subcutaneously along with 1 mg of gallamine triethiodide (muscle relaxant). The mean heart rate in embryos was 287 ± 24 bpm (n = 1,013). Embryos with heart rates <215 bpm were excluded from study. Brain temperatures of embryos younger than stage 42 was maintained at a mean of 35 ± 2.39°C (n = 85), whereas in older animals it was 37.2 ± 1.6°C (n = 1,006).
Posthatch birds were anesthetized using an intramuscular injection of EquiThesin (0.003 ml/g) and gallamine triethiodide (1 mg) and given hourly supplements of EquiThesin (0.05 ml) to maintain anesthetic level. The lungs were perfused with an oxygen-enriched, humidified warm air/CO2 mixture as described in detail elsewhere (Nazareth and Jones 1998). Brain temperature for hatchlings was maintained at 38.4 ± 1.6°C (n = 209) and heart rate was 458 ± 23 bpm (n = 209). The University of Missouri Institutional Animal Care and Use Committee approved the care and use of the animals described herein. In all cases the work was carried out in adherence to The American Physiological Society's Guiding Principles in the Care and Use of Animals.
In all animals, the beaks were embedded in plaster to stabilize the head in a position with beak down. The nasooccipital axis of the head was adjusted nearly 30° off vertical to the right and posterior. A small opening was made through the bony plate overlying the recessus scala tympani and the periosteal lining of labyrinth was opened to expose the underlying perilymph. Glass micropipettes were filled with 0.5 M KCl and 0.05 M Tris (pH 7.4). The electrometer (WPI Intra 767) provided for current injection and periodic impedance checks (20–100 M
). Reference (neck) and ground (thorax for posthatch birds, extraembryonic fluid for embryos) electrodes were chlorided-silver wire. A Burleigh inchworm stepper was used to position microelectrodes. Recordings were made of isolated single cochlear primary afferent neurons (cochlear ganglion cell bodies or their processes). Auditory neural activity was amplified, led to a window discriminator, a spike timer, and an analog tape recorder for storage and off-line analysis.
Stimulation and response measurements
Airborne sound stimuli were delivered using a calibrated Etymotic ER2 earphone inserted and sealed into place in the left external auditory meatus (EAM). This method of sound presentation is referred to as "airborne" stimulation throughout this report. Sound levels were measured in dB SPL re: 20 µPa and the maximum stimulus level available was about 100 dB SPL. A calibrated probe tube microphone (Etymotic ER7) was sealed in the EAM and used to monitor the SPL near the tympanic membrane. Clicks, pure tones, noise, or pure-tone bursts were used as stimuli to determine whether individual cells responded to sound. Pure-tone bursts (i.e., 5-ms onset/offset ramp, a range of plateau durations from 20 to 80 ms, 50 to 6,000 Hz) were used to estimate the frequency eliciting the maximum level of firing. In most cases, an automated procedure [termed quick tune (QT)] was used to make a rapid estimate of the frequency producing a maximum response as follows. The response of the cell was recorded continuously during the presentation of a constant-amplitude sine-wave frequency sweep (generally 100 to 5,000 Hz). The frequency generating the highest spike rates was defined as the "best frequency" and designated here as CF* to distinguish it from the traditional CF defined below.
A computerized threshold-tracking procedure was completed to obtain an FTC. Response threshold was determined for each frequency [typically 50 frequencies from 100 to 5,000 Hz; tone bursts: 5-ms rise/fall time; plateau of 40 ms (hatchlings) or 80 ms (embryos)]. The number of spikes that occurred during the stimulus plateau and the number occurring during an equal period of silence (i.e., no stimulus) were subtracted. A difference of two spikes was set as the criterion for response threshold at each frequency. The CF for each FTC was documented and defined as the frequency corresponding to the lowest threshold level.
Poststimulus time histograms (PSTHs) were also generated. The onset time for each spike was logged during a specified period (usually 200 ms) immediately after the onset of a tone burst stimulus. This was repeated to accumulate spike counts in time bins (commonly 1–5 ms). When possible, a rate–intensity matrix was determined for a cell using 50 frequencies and 12 stimulus levels, with each combination presented in random order. All FTCs and PSTHs were measured on-line and spike timing recorded at a conversion rate of 1 µs per point. Spontaneous discharge activity was recorded on tape and analyzed off-line.
Driving the columella footplate directly
A piezoelectric (PZL) driver (Burleigh PZL-007-20) was configured with a stainless steel tube extension (OD 
1 mm; Fig. 1), calibrated (see following text), and used to produce sinusoidal displacements (vibrations) of the columella footplate. This device was used to apply a vibration directly to the footplate and thus bypass the middle ear conduction apparatus. A sine-wave input signal was used to control the amplitude and frequency of the driver output. This method of stimulation is referred to throughout the text as footplate stimulation. The velocity of the driver tip, v(t), was calculated based on the following equation, where Ap is peak displacement, 
= 2
f, f is frequency, and displacement expressed as a function of time is Ap sin t
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). Coupling of the PZL driver tip to the columella footplate was accomplished by removing the tympanic membrane and carefully severing the columella (using microsurgical spring scissors) to leave a 1- to 2-mm stump attached to the footplate. Care was taken to minimize motion of the footplate with the cut and to avoid contact between the tip of the scissors and the nearby round window. Figure 1 illustrates the placement of the tip of the driver tube (Pu). The driver tube was slipped over the columella stump and advanced by micropositioner to rest against the outer surface of the footplate (FP). Once in contact with the surface of the footplate, the tip was slowly advanced 50 to 100 microns to place the driver tip–footplate junction under slight restoring elastic tension. This provided a secure mechanical coupling between the tip and footplate such that the two moved in unison during tip motion. Submicron displacement amplitudes were used during stimulation and the maximum possible sinusoidal displacement (although not used in these studies) was on the order of 2 microns peak-to-peak (µmp–p). The cochlear microphonic was recorded from the scala tympani and used to monitor the effectiveness of the airborne and footplate stimulation as well as the overall physiological status of the ear.
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Tip motion was calibrated as particle velocity in units of millimeters per second. We represent tip particle velocity in terms of its kinetic equivalent in air. That is, PZL tip velocity was expressed as the sound pressure level in air that produces an equivalent air particle velocity and is designated as dB keSPL here. Kinetic equivalent dB SPL is the sound pressure level (dB SPL) that corresponds to a pressure satisfying the equation: Prms = VrmsZair, where Vrms is the particle velocity of the PZL tip and Zair is the specific acoustic impedance of air (0.4145 Pa · s/mm; see following text). In our view, the use of dB keSPL facilitates the comparison of stimulus levels for footplate stimulation and traditional airborne stimulation. Kinetic equivalent levels can be thought of as the airborne stimulus level in dB SPL that would reproduce a given footplate particle velocity if the middle ear provided a perfect impedance match and thus accomplished a perfect transfer of particle velocities of airborne sound to the footplate.
PZL driver tip particle velocity can be readily calculated from dB keSPL. The following equations may be used to convert between these representations. Sound pressure [in pascals (Pa)] is proportional to the particle velocity (mm/s) and is given by Prms = VrmsZair as defined in the preceding paragraph. The characteristic impedance of air at 20°C and 760 mmHg is given by Zair = 
c, where is the density of air and c is the speed of sound in air. Taking as 1.205 (kg/m3) and c as 344 (m/s), then it follows that Zair = 0.4145 Pa · s/mm. Particle velocity (mm/s) is thus Vrms = Prms/0.4145. The sound pressure in pascals can be determined for any value of dB SPL and particle velocity can be calculated as follows: Vrms = {0.00002[10(X/20)]}/0.4145, where X is the sound pressure level of interest in dB SPL (for air) or dB keSPL (for PZL tip) and 0.00002 is the standard 20 µParms reference for dB SPL. Because the tip particle velocity of the PZL driver is known and is the same as the footplate during stimulation, then the kinetic equivalent sound pressure level can be calculated as follows: X = 20 log [(VptrmsZair)/0.00002], where Vptrms is the rms particle velocity of the PZL driver tip and X is expressed in dB keSPL.
Estimating middle ear transfer effects
Mechanically driving the columella footplate directly makes it possible to precisely control the amplitude of sound vibrations entering the cochlea. By noting the difference between the sound pressure levels of airborne stimuli required to achieve a given footplate velocity and the corresponding dB keSPL for the same velocity we can estimate the transfer loss in decibels. Because the response threshold of auditory neurons depends on the particle velocity of sound entering the cochlea (principally by the footplate), it follows that collective differences in thresholds to airborne stimuli (dB SPL) and footplate stimulation (dB keSPL) provide indirect estimates of transfer losses from air to the cochlea. These differences are noted here as conductive losses.
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RESULTS |
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We recorded a total of 1,282 neurons, 1,046 in embryos and 236 in hatchlings. Of these, we were able to determine whether 949 embryonic and 227 hatchling cells responded to sound. An estimate of CF* was made for 516 embryonic and 125 hatchling units, whereas a CF was obtained from the FTCs of 181 embryonic and 147 hatchling units. Another 70 cells isolated in young embryos, although responsive to sound, exhibited FTCs that were flat or obscured by noisy threshold curves, thus precluding a definitive CF assignment. Both CF* and CF were obtained for many cells in embryos and hatchlings and a comparison of these data indicated that CF* was a reasonable estimate of CF in the present study. Regressions of CF* on CF were highly significant (P < 0.001) with slopes near 1.0 (embryos: slope = 0.939, intercept = –31.9 Hz, P < 0.001, R2 = 0.96; hatchlings: slope = 0.956, intercept = 43.4 Hz, P < 0.001, R2 = 0.98).
The emergence of cochlear primary afferent responses to airborne sound
INCIPIENT COCHLEAR AFFERENT NEURAL RESPONSES. Incipient responses to airborne sound were simple and sluggish (i.e., action potential spikes were poorly timed to stimulus as well as few and inconsistent in number). The earliest appearance of such responses occurred at late stages 39 to late 40 (39+ to 40+, E14). Most cells were unresponsive to sound at levels <100 dB SPL at this age (i.e., 87% unresponsive, total sample = ntot = 30). Simple primary afferent responses to airborne sound occurred more frequently through stages 40+ and 41 yet the majority remained unresponsive (i.e., 63% unresponsive, ntot = 38). Although we did not evaluate large numbers of cells at stages from 39 to 41+, those that did respond to sound were very insensitive to stimuli (73% unresponsive, ntot = 66). Thresholds were determined in 12 of these cells and the mean value was 89.2 ± 6.5 dB SPL (n = 12). These cells demonstrated no evidence of appreciable frequency selectivity (no tuning), responded to a wide range of frequencies, and evidenced a relatively flat high-threshold response curve (>90 dB SPL; Figs. 2 and 3). The early rudimentary response period extended beyond stage 41 into stage 42 in some animals. The cumulative proportion of cells that were unresponsive to airborne stimuli remained high through stage 42+ (73%, ntot = 113). Figure 2 illustrates responses from several neurons at stages between 40 and 42, including one cell (top: 193tm05) responding best to 3,000 Hz (threshold 102 dB SPL). The bottom panels of Fig. 2 show PSTHs obtained at these stages. A number of important features of afferent responses can be discerned. Cells, although profoundly insensitive, could respond to a broad range of frequencies, including high frequencies. Individual cell response patterns were variable, of late onset after the stimulus onset, and could fail altogether. Airborne stimuli were used for all examples shown in Figs. 2 and 3. The top right histogram of Fig. 2 (193tm05) reflects responses of late onset but its overall shape approaches most closely that of a mature PSTH. Figure 3 (e.g., labels "41") provides examples of "flat" threshold curves obtained from stage 41 ganglion neurons. The top four curves of Fig. 3 illustrate the typical high-threshold nature of curves and the absence of appreciable frequency selectivity for incipient responses.
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Maturation of frequency selectivity occurred rapidly and dramatically after stage 42. Figure 4 illustrates the overall progression of change in FTCs proceeding from early to late embryonic stages (i.e., 41 to 43) using examples having CFs occurring near 1,000 Hz. Figure 5 summarizes the distribution of CFs and CF thresholds for each cell recorded across stages 39 to 44 using airborne stimulation. Note that CF threshold levels for cell stages 39+ to 42+ were relatively high for airborne stimuli [mean = 86.0 ± 10.9 dB SPL (n = 26)] and CFs varied widely (range: 305 to 2,841 Hz, Fig. 5) as a result of the relatively flat FTCs observed at the earliest ages (i.e., essentially no tuning; Fig. 3). As frequency selectivity became more pronounced, lowest threshold regions of FTCs became sharper and began to form tips (Fig. 3, bottom four curves). CF threshold levels for airborne stimulation decreased dramatically in the older embryos [e.g., Fig. 4 and Fig. 5 group legend: "43 to 44"; mean = 60.5 ± 18.2 dB SPL (n = 24)] and CFs became restricted to frequencies 
2,300 Hz.
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FTCs for embryos and hatchlings using both airborne and footplate stimulation are shown in Fig. 7. The FTCs produced using both methods were generally comparable in the late embryo and posthatch animal as can be seen from the figure.
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For those cells that responded to PZL footplate driving through stage 42+ and where thresholds could be estimated, the threshold level required to activate the cells was on average 43.3 ± 9.5 dB keSPL (n = 14) and this was considerably less than that required for airborne sound during the same period (as given above: 86.0 dB SPL). The lowest threshold level for footplate stimulation for this age group was 31.4 dB keSPL.
Figure 6 (middle and bottom) summarizes raw (middle) and mean (bottom) CF threshold data for footplate stimulation. There was a negative correlation between CF threshold level and embryonic stage for footplate stimulation (regression slope = –6.162 dB/stage; P = 0.001; R2 = 0.07). The effect size (Cohen 1988) of stage on threshold (R = 0.26) was relatively small for footplate stimulation, where the proportion of variance attributable to age was <10%. This is considerably smaller than the effect size of embryonic stage on threshold for airborne stimuli (where R = 0.55, R2 = 0.31, and the proportion of variation 30%).
EMERGENCE OF FREQUENCY SELECTIVITY IN RESPONSES TO FOOTPLATE STIMULATION. Studies using the PZL driver also confirmed the developmental period where frequency selectivity began to appear. We have documented FTCs for a small number of cells in the early embryo. The shape of FTCs at stages 41+ to 42+ obtained with footplate stimulation often had an unusually flat low-frequency (LF) tail combined with a steep high-frequency (HF) slope. Several examples of FTCs in early embryos are illustrated in Fig. 8. None of the detailed footplate FTCs in early embryos took on the mature "V" shape of older animals. Most cells responded preferentially to low-frequency stimuli but there were examples of cells that responded well to much higher frequencies. For early periods between stages 41 and 42+, complete FTCs were available for only a few cells. Figure 5 displays all CF thresholds successfully recorded from embryos younger than stage 43 (direct foot plate and airborne stimulation). The range of frequencies for footplate stimulation was 245 to 1,497 Hz (n = 14) for stages 41 to 42+. There was no systematic attempt to adjust tip position toward the base or any other particular cochlear region in these youngest animals, and thus no assumption regarding the position of cochlear primary afferent terminal fields can be made for recorded neurons.
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3,500 Hz, it was necessary to reposition the animal and to direct the microelectrode tip toward more basal regions of the ganglion. This required some effort especially for the highest CFs >4,000 Hz. This added procedural difficulty presumably arose from the fact that there is a natural barrier, noted by others (Manley 1990; Manley et al. 1985, 1991; Salvi et al. 1992), that reduces access to the extreme basal regions of the ganglion. Adjustments in the electrode path were effective in mitigating this problem. Figure 9 summarizes the overall distribution of CFs (n = 181) and CF*s in the embryo (n = 516, CF*).
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). A second feature of FTCs from embryos was the appearance of shallow LF slopes in comparison to those of hatchlings. This also can be seen in Fig. 11 where it is most prominent in the FTCs with CFs >4,000 Hz.
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During stages 43 and 44 the overall mean CF threshold levels for airborne stimulation fell to their lowest values as indicated in Fig. 6 (top). Collectively, airborne CF threshold levels for stages >42.5 had a mean value of 60.5 ± 18.2 dB SPL (n = 24) compared with stages <43, where the mean threshold level was 86 dB SPL as stated above. Thus the difference between the two embryonic periods (early: <43, vs. late: >42.5) was on the order of 26 dB SPL. In contrast, the difference in mean threshold levels between the same two periods of development for footplate stimulation was not remarkable [Fig. 6, early embryos: 43.3 ± 9.5 dB keSPL (n = 14); late embryos: 41.8 ± 14.7 dB keSPL (n = 133)]. This is consistent with the smaller effect size shown for the correlation of age and thresholds for footplate stimulation (i.e., R = 0.26). These findings also account for the observation that the difference in mean airborne and footplate threshold levels for the early embryos (stages <43) was 42.7 dB, whereas the difference for the oldest embryos (stages >42.5) was 18.7 dB.
Despite the small effects of age on mean thresholds for footplate stimulation, there were important changes in the range of CF thresholds for footplate stimulation in late embryos. During stages 43 to 44, the most sensitive individual CF thresholds for footplate stimulation were obtained (Fig. 6). Indeed the lowest CF threshold level overall was found during this late period (5 dB keSPL) and the lowest values across CF frequencies ranged from 5 to 25 dB keSPL (Fig. 12, top). Thus the appearance of the lowest CF threshold levels occurred in concert with improved frequency selectivity in the late embryo (Figs. 6 and 12).
Some general trends are suggested by the distribution of CFs and CF thresholds in Fig. 12. A dotted line has been drawn in Fig. 12 that marks out the contour formed by the lowest individual CF threshold levels across CF frequencies for embryos. The shape is reminiscent of an audibility curve. The line represents the approximate boundary of lowest response threshold levels for the late embryonic cochlea when the middle ear was bypassed and the cochlea was stimulated by directly vibrating the columella footplate. Following the line contour across frequencies from 200 to 2,000 Hz, the lowest CF threshold levels tended to drop sharply by about 40 dB to values <20 dB keSPL. Above 2,000 Hz, the lowest CF threshold levels increased again to range from 30 to 50 dB at frequencies >3,000 Hz. Most cells of late embryos had CF thresholds between 20 and 60 dB keSPL.
Although the mean CF threshold level for footplate stimulation was about 42 dB keSPL in the late embryo, eight of the cells had threshold levels between 10 and 20 dB keSPL with CFs between about 800 and 3,000 Hz. The mean CF threshold level for values <20 dB keSPL in the late embryo was 13.7 ± 3.9 dB keSPL (n = 9). The lowest CF threshold level was 5 dB keSPL with CF at 1,706 Hz. If we take this lowest CF threshold level as a lower limit of audibility, we can estimate the lowest cochlear threshold particle velocity and displacement obtained in the late chicken embryo. This value corresponded to a peak velocity of about 8.58 µm/s and a peak displacement of about 0.8 nm. Clearly, based on these data, when the middle ear was bypassed, there was no obvious major systematic restriction in primary afferent threshold sensitivities over the frequency range from 100 to 4,500 Hz.
Frequency selectivity and CF thresholds in hatchlings
The results of measuring FTCs, CFs, and CF thresholds in posthatch animals are shown in Figs. 6, 9, and 12. The overall distribution of CFs (n = 147) and CF*s (n = 125) are shown in Fig. 9 (top). Although there were fewer cells completed for the hatchling, the overall distribution of CFs and CF*s provided a useful comparison to the embryo.
When CF threshold levels of embryos were compared directly to posthatch animals, both age (embryo vs. posthatch) and stimulus type (airborne vs. footplate stimulation) had significant effects [two-factor ANOVA, P < 0.001, F = 74.7, degrees of freedom (df) = 3]. CF threshold levels were significantly lower in posthatch animals compared with embryos (two-factor ANOVA, P < 0.001, F = 122.2, df = 1), whereas CF threshold levels for footplate stimulation were significantly lower than the levels for airborne stimulation (two-factor ANOVA, P < 0.001, F = 74.6, df = 1). However, the lower threshold levels for footplate stimulation were found only in embryos and the lower threshold levels for posthatch animals were found only for airborne stimulation (two-factor ANOVA interaction, P < 0.001, F = 99.5, df = 1). These relationships are best illustrated in Fig. 6, which shows individual and mean CF threshold levels for each stage of development. On average, airborne CF threshold levels for hatchlings were 22.6 dB SPL lower than those of late embryos (stages >42.5). CF threshold levels for airborne and footplate stimulation had considerable overlap in hatchlings (Figs. 6 and 12) and mean values reflected this [airborne mean = 37.90 ± 12.6 dB SPL (n = 79); footplate mean = 40.1 ± 12.2 dB keSPL (n = 68)]. Mean threshold levels in hatchlings were the same as those of late embryos with footplate stimulation (mean as given above = 41.8 dB keSPL).
One other obvious difference from embryos was the unrestricted range of airborne CFs displayed by the posthatch animals. Figure 12 (bottom) illustrates airborne CFs from 200 to 4,717 Hz for the hatchling, whereas the highest airborne CF for embryos of Fig. 12 (top) was 2,175 Hz. Remarkably, however, the range of CFs for footplate stimulation in the embryo was similar to that for hatchlings.
The CF threshold distribution for airborne stimulation in hatchlings found here was comparable to those reported elsewhere (Jones and Jones 2000; e.g., Manley et al. 1985, 1991; Plontke et al. 1999; Saunders et al. 1996). The dotted contour line bounding the most sensitive threshold limits for embryos Fig. 12 (top) is reproduced in Fig. 12 (bottom) to facilitate comparison. The line accentuates the fact that there were fewer CF threshold levels <20 dB keSPL for footplate stimulation in hatchlings compared with embryos. This is likely attributable to the smaller sample obtained for hatchlings. At CFs near 200 Hz, the lowest CF thresholds were about 60 dB SPL/keSPL. The lowest CF thresholds then decreased by 20 to 40 dB as CF approached 1,000 Hz. The lowest CF thresholds increased sharply again as CF increased to >3,000 Hz.
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DISCUSSION |
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In the present study, we evaluated the emergence of cochlear primary afferent responses to sound during development of hearing in the chicken embryo. Most cochlear ganglion neurons did not respond to sound levels <100 dB SPL at stages 39 to 42 and those few that did respond had high-threshold levels generally >80–90 dB SPL. Under most natural circumstances, this altogether precludes the detection of ambient environmental sound. Indeed, this early period of profound insensitivity continued at least through stage 41 and into 42 and encompassed incubation days at least as early as E12 and extended well into E16. We have concluded therefore that this period of development is essentially prehearing. During this period a large proportion of cells were unresponsive even when the middle ear was bypassed during footplate stimulation. However, the proportion of unresponsive cells was considerably reduced and the mean threshold level for cells that did respond to footplate driving was significantly below (about 40 dB) that of cells responding to airborne stimuli in age-matched embryos (stages <43; e.g., see Fig. 6). These findings indicate that the insensitivity of cochlear ganglion neurons to airborne sounds arose in part from middle ear transfer immaturities and that the average magnitude of the conductive loss was on the order of 40 dB.
Reduced transfer of airborne sound to the cochlea alone, however, cannot account for CF threshold levels in excess of 80 dB SPL in embryos younger than stage 43. Indeed, the cochlear apparatus itself was less sensitive to footplate stimulation at these younger ages because cochlear ganglion neurons often failed to respond at all levels. Moreover, the lowest CF threshold levels for footplate stimulation decrease about 20 dB by stages >42.5. Thus changes in the sensitivity of the cochlear apparatus itself contribute to early maturation.
The onset of frequency selectivity
Before E16, cochlear primary afferent neurons do not likely encode frequency information about the natural sound environment. From the present findings, one can place the emergence of frequency selectivity in cochlear ganglion neurons between stage 41 and 42. Even so, initial response preferences for certain frequencies were rudimentary and threshold levels for airborne stimuli were generally 80 dB SPL (Fig. 3). When the footplate was driven directly, neural FTCs generally emerged slightly earlier (early E16, 41+) with steep HF slopes and flat or very shallow LF tails. The flat LF tails of FTCs indicated an increased broad sensitivity to low-frequency stimuli below CF. The general shape of the early FTCs is reminiscent of a low-pass filter with a steep HF roll off. This feature of early response patterns may contribute to mechanisms responsible for the well-known increased responsiveness to low-frequency sounds exhibited by young embryos. The flat or shallow LF tail of incipient FTCs almost certainly would lead to manual audiovisual CF estimates biased toward the LF tail. Steep LF slopes develop in FTCs almost immediately thereafter such that relatively mature slopes are expressed at stages 43 and 44 (contrast Figs. 8 and 10).
The basis for the rapid transformation from flat to steep LF slopes is not clear. Based on the findings we can suggest that mechanisms within the cochlea rather than sound transfer changes are responsible for the transition to steep LF slopes. There is evidence that mechanical tuning on the avian papilla may be influenced by electrical resonance properties of hair cells (Duncan and Fuchs 2003; Fettiplace and Fuchs 1999; Manley 2001). These investigators suggested that large-conductance calcium-activated potassium (BK) ion channels, normally resident in the short hair cells of the mature papilla, are key mediators of electrical resonance properties. One interesting aspect of this hypothesis is the developmental time course for the appearance of BK channels in hair cells. Remarkably, it coincides with the onset and maturation of frequency selectivity in primary afferent neurons reported here. Although intriguing, evidence providing a direct linkage between FTC maturation and BK channel activity in the bird is not available.
Frequency selectivity in the late embryo
Most FTCs in the late embryo (i.e., stage 43 to 44) had mature characteristics including steep high- and low-frequency slopes as illustrated in Fig. 10. Although there was considerable variability in maturity across animals at any given age, there were consistently a few lingering immature features in many FTCs of the late embryo. These included "jagged" or "sawtooth" curves (described in Jones and Jones 1995a) and shallow LF tails. Whereas the "jagged" contours appeared to be independent of CF, the LF shallow tail seemed to be a common feature in high-frequency tuning curves (i.e., CFs >2,000 Hz) even in the late embryo (Fig. 11). These features may be remnant immaturities reflecting the final stages of refinements in frequency selectivity.
The range of CFs in the late embryo
One of the most striking differences between embryonic FTCs obtained using airborne stimuli and those found during footplate stimulation was the range of CFs observed. The CF distribution for airborne stimulation in the late embryo herein is comparable with that reported earlier (Jones and Jones 1995a), i.e., a restricted CF range 2,000 Hz. The absence of CFs substantially >2,000 Hz (i.e., 2,200 to 5,000 Hz) for airborne stimulation in the late embryo underscores one of the central questions addressed by this research program. Figures 9, 10, 11, and 12 (top) demonstrate that the late embryonic papilla actually mediates a wide adultlike distribution of FTCs when the footplate is driven directly.
A description of tuning curves from auditory neurons with CFs >4,000 Hz in the chicken is remarkable even for posthatch animals, inasmuch as there have been only a handful of cells with such CFs reported in the literature. Examples of the highest CFs are 4,956 Hz in the adult cochlea (Chen et al. 1996), 4,545 Hz in the hatchling cochlea (Jones and Jones 2000), 4,840 Hz in the hatchling brain stem (Warchol and Dallos 1990), and 5,000 Hz in the adult mesencephalon (Coles and Aitkin 1979). The finding of CFs well above 3,000 Hz in the embryo was surprising to us because our working hypothesis at the start of these investigations was that the base did not function at all. The discovery of CFs with frequencies ranging from 2,200 to 4,500 Hz establishes that the base is functional in the late embryo. This applies to embryonic ages as young as E17 and E18 (stages 43 and 44). One may conclude that the primary afferent neurons generating FTCs with CFs >2,000 Hz innervate regions occupying approximately the basal 40% of the papilla because frequencies below this have been shown to be mediated by the apical 60% in the late embryo (Jones and Jones 1995b). The present results confirm and extend our earlier findings (Jones and Jones 1995b) and are also consistent with the findings in the chicken at other ages (Chen et al. 1994; Manley 1996; Manley et al. 1987; Von Békésy 1960).
A comment on the shifting tonotopic map hypothesis
The first key element of the well-known shifting tonotopic map hypothesis (Lippe and Rubel 1983, 1985; Rubel and Ryals 1983; Rubel et al. 1976, 1984) required in the late embryonic chicken is that the cochlear base mediates the first responses to sound and responds best to low frequencies (e.g., 100–2,000 Hz). Simultaneously, the hypothesis required that the apex be immature and nonfunctional. A second key element was that the map was dynamic, whereby regions mediating low frequencies shifted from the base to the apex and, in the region of the base itself, best frequencies shifted to increasingly higher values. The size of the putative frequency shifts from late embryo (stage 43) to hatchling was reportedly on the order of an octave or more and the mature adult audibility range of 100 to 4,100 Hz (Rubel and Parks 1975) was not achieved until P14 and beyond. Subsequent investigations in the chicken did not uniformly support the hypothesis (Cotanche et al. 1987; Cousillas and Rebillard 1985; Jones and Jones 1995b; Manley et al. 1987; see review see Manley 1996) suggesting to the contrary that there was no evidence of a base-to-apex shifting tonotopic map over the ages originally proposed for such shifts.
Despite evidence to the contrary in the chicken, a number of investigators (e.g., Romand 1997; Rübsamen and Lippe 1998) continued to entertain the possibility of shifts in the late embryo. For example, Rubsamen and Lippe (1998) indicated that there may yet be a map shift at earlier embryonic periods (i.e., at E17, the earliest age where changes in central maps were reported). In the present study, it was demonstrated with direct measurements at E17 and E18 (stages 43 and 44) that primary afferents already mediate the full adult range of audibility in the chicken. One logical conclusion that can be drawn from this finding is that an octave shift in cochlear frequency is clearly not required to achieve an adult CF distribution in the cochlea. Stated simply, there is no need to invoke a shifting map.
Restricted high-frequency CFs with airborne sound stimulation
The findings here of CFs as high as 4,478 Hz requires that the base functions sufficiently to produce such FTCs. Given this fact, why were such CFs not found in earlier studies? A number of explanations for the restricted CFs have been offered in the past (e.g., Jones 1994; Jones and Jones 1995b; Manley 1990, 1996; Manley et al. 1985, 1991). It has been argued that 1) the basal 30–40% of the cochlea may be nonfunctional, 2) there may be a restricted transfer of airborne high-frequency sound to the cochlea in the late embryo, 3) there may be a sampling bias for low- to middle-frequency neurons, or 4) some combination of these alternatives is responsible for the absence of high CFs.
We can rule out the first alternative (i.e., the base does not work) because we would not have observed high-frequency CFs in this case with footplate stimulation. Our results support the last two hypotheses: there is a high-frequency transfer loss of airborne sound and there is a systematic sampling bias against high-frequency neurons. We report here that a remarkable increase in the number of CFs well above 2,000 Hz occurred when footplate stimulation was used, yet the highest CFs well above 3,000 Hz were obtained only after an adjustment in the electrode path was made. The success of the readjustment (i.e., CFs approaching 4,500 Hz were obtained) explicitly demonstrated that a sampling bias does normally operate when accessing the ganglion from the recessus scala tympani. These results account for the difficulty typically found in isolating neurons with CFs well above 3,000 Hz. A similar improvement in the number of high-frequency CFs was reported by Chen et al. (1996; compare with Salvi et al. 1992).
A structural barrier alone does not account for the marked improvement in the number of high-frequency CFs found without readjusting the electrode path during footplate stimulation. Greater numbers of high CFs in this case could arise only from an improvement in delivery of high-frequency sound to the cochlea with footplate stimulation. This finding indicates that, in the embryo normally, high-frequency sound transfer is dramatically and preferentially restricted. Footplate driving eliminates the conductive loss at high frequencies. Together, adjusted electrode path and footplate stimulation reveal a fully mature range of CFs. Saunders and colleagues (1973) hypothesized many years ago that the middle ear played an important role in reducing sound transfer at high frequencies. Our findings confirm this proposal.
CF threshold distributions for embryos and hatchlings
The mean CF threshold level for airborne stimulation was significantly higher for embryos than for hatchlings (Figs. 6 and 12), whereas there was no difference in mean threshold levels for footplate stimulation for the two ages. These results, the considerations above, and findings from other laboratories (Cohen et al. 1992; Cotanche et al. 1987; Gates et al. 1975; Kim 2002; Saunders 1985; Saunders et al. 1986) provide clear evidence for general improvements in the sound transfer characteristics of the middle ear at the time of hatch and several weeks thereafter. The threshold improvements appear to arise without a dramatic parallel change in the sensitivity of the cochlea itself.
We found differences between airborne and footplate CF threshold levels in embryos that decreased with stage. Such findings were indicative of important general improvements (i.e., across a broad range of CFs) in sound transfer during embryonic stages as well.
The emergence of hearing in the chicken embryo
It is appropriate to integrate the present findings with recent reports from our laboratory as well as a long-established literature. The aim is to clarify and summarize the timeline of major functional milestones in the ontogeny of hearing in light of the new information. We offer a synthesis toward a broader view and a working hypothesis for the ontogeny of hearing in the chicken.
Perhaps the most fundamental adjustment to be made in how one views the cochlea and hearing in the chicken is to realize that the cochlea is not always a sensor during ontogeny. Synaptic contacts between hair cells and afferents appear (E8 to E11; Cohen and Fermin 1978; Hirokawa 1978; Rebillard and Pujol 1983; Whitehead and Morest 1985a,b) and central synapses between primary afferents and second-order cells of NM are functional (
E12; Jackson et al. 1982; Pettigrew et al. 1988) by about E12. However, it is apparent from the present findings that detecting and processing of natural ambient sound through the level of primary afferents by the cochlea cannot occur to any appreciable extent until days after this. We suggest that hearing must thus begin much later. We argue that the "prehearing" period extends at least to late E16 when threshold levels for airborne sounds fall generally well below 90 dB SPL. A similar prehearing period has been appreciated in neonatal mammals for some time and is commonly regarded as a delay period in hearing onset after synaptogenesis.
The cochlea is active and does signal to brain stem pathways during embryonic prehearing periods (i.e., period of "Endogenous Signaling"; Fig. 13). Cochlear ganglion neurons develop endogenous discharge patterns beginning by stage 39 (E13; Jones et al. 2001). The activity patterns are spontaneous, rhythmic, and fundamentally different from those of ganglion neurons in the mature cochlea. The unusual rhythms are present from about E12 through E17 (Jones et al. 2001). It is likely that these discharge patterns activate central auditory relays (Jackson et al. 1982; Pettigrew et al. 1988) and therefore present a major influence on central cells and the refinements of neural pathways there. Spontaneous central rhythmic activity is also present at least as early as E14 through E17, a period overlapping similar discharge patterns in the cochlea and these central rhythmic patterns depend on eighth nerve discharge activity (Lippe 1994). All of these findings support the hypothesis that ganglion cells of the cochlea are the pattern generators for central rhythmic discharge from stage 39 through stage 43.
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; Parks 1979; see review by Rubel and Fritzsch 2002). However, the distinction between spontaneous and stimulus-dependent auditory nerve activity during this period is consistently blurred in the literature. We have found that primary afferents are unresponsive to natural ambient sound levels at these early stages. We thus hypothesize that spontaneous discharge patterns of primary afferents alone provide the activity-dependent neurotrophic action on central auditory relays during prehearing periods in the chicken (E12 to E16, stages 39 to 42+). There is speculation that these spontaneous rhythmic discharge patterns play more specific roles as well, including a role supporting Hebbian processes (Hebb 1949) associated with central synaptic refinements (Jones et al. 2001; Lippe 1994).
The time line for cochlear ontogeny therefore begins with endogenous rhythms around the period of peripheral synaptogenesis or soon thereafter as illustrated in Fig. 13. Responses to natural ambient sound levels begin to emerge much later, around stages 41 to 42, and frequency selectivity subsequent to that (Fig. 13, "Detecting and Encoding Sound" and "Tuning"). Spontaneous activity begins to change during periods where responses to sound emerge (Jones et al. 2001). Rhythmic bursting patterns are replaced over a period of several days by patterns of irregular bursting (stages 41 to 44) and ultimately by quasi-Poisson discharge patterns of the mature animal. From stages 42+ to 44, emergent tuning curves with flat LF tails are replaced with the more familiar mature "V-shaped" FTCs along with the appearance of a mature complement of CFs ranging from about 50 to 5,000 Hz.
Dramatic changes in sound transfer from air to the cochlea as well as in the sensitivity of the cochlea to footplate stimulation occur during the maturation of cochlear frequency selectivity. Our findings do not indicate that there are major changes in cochlear sensitivity immediately before and after hatch. On the other hand, we can confirm substantial decreases in the impedance to sound transfer from air to the cochlea between the late embryo and 2 wk posthatch. Thus even in the late embryo (stage 44, E18–E19), there remain important restrictions on the transfer of sound, particularly high-frequency sounds, to the cochlea. The present results also confirm that improvements in behavioral thresholds immediately after hatch (Gray and Rubel 1985) occur primarily as a result of improved sound transfer to the cochlea as previously hypothesized by others (Cohen et al. 1992; Cotanche et al. 1987; Gates et al. 1975; Saunders et al. 1986).
Summary working hypothesis
On the basis of our findings we offer a working hypothesis that underscores two major developmental periods. First is a period of endogenous cochlear signaling that is critical for neurotrophic support of central relay nuclei and for guiding normal developmental refinements in central binaural processing pathways. Second is a later period where the cochlear sensor acquires the ability to perform ambient sound detection and encoding. Hypothetically, the natural restriction of ambient sound transfer during periods of endogenous signaling may be a key requirement for refinements in central processing tracts and normal development. The cochlea itself begins to mature and acquire the ability to transduce and encode the features of sound before major improvements occur in ambient sound transfer to the cochlea. The late embryo presents a cochlea with nearly mature frequency selectivity and sensitivity in combination with a low-pass sound transfer system, where a substantial high-frequency conductive loss remains in place. A dramatic change occurs in the neonate and by 2 wk posthatch: frequency selectivity and sensitivities to ambient sound approach those for the adult across all frequencies. This transformation is dominated by changes in the transfer characteristics of the middle ear because the range of CFs present as well as threshold levels for responses to footplate stimulation do not appreciably change during the same period (i.e., embryo to hatchling).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: T. A. Jones, Department of Communication Sciences and Disorders (CSDI), School of Allied Health Sciences, East Carolina University, Belk Annex, Charles and Greenville Blvds, Greenville, NC 27858 (E-mail: Jonesti{at}ecu.edu)
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INTRODUCTION
