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6J mice studied with auditory evoked response tuning curves
Brain Research, 187 (1980) 69-79 © Elsevier/North-Holland Biomedical Press
69
T H E M A T U R A T I O N OF F R E Q U E N C Y SELECTIVITY IN C57BL/6J MICE S T U D I E D W I T H A U D I T O R Y EVOKED RESPONSE T U N I N G CURVES
JAMES C. SAUNDERS, KIM G. DOLGIN and LOUIS D. LOWRY Department of Otorhinolaryngology and Human Communication and Department of Psychology, University of Pennsylvania, Philadelphia, Pa, 19104 (U.S.A.)
(Accepted August 30th, 1979) Key words: frequency selectivity - - tuning curves - - auditory development - - cochlear nucleus
SUMMARY The present study was designed to examine the ontogeny of frequency selectivity in the neonatal auditory system. Mice were tested between 12 and 65 days of age. At each age two measures of auditory sensitivity were made from cochlear nucleus evoked responses. Tone-burst evoked-response thresholds in the quiet were determined for frequencies between 1.0 and 39.0 kHz. A two-tone simultaneous masking procedure was then used to obtain evoked response tuning curves. The frequency selectivity of the tuning curves was quantified by calculating a Q ratio. The results show that tuning is poor in neonates but rapidly improves to adult-like levels within 5-16 days after the inception of auditory function. The data also indicate that the development of frequency selectivity varies directly with the maturation of threshold sensitivity.
INTRODUCTION In his recent review of structural and functional developments within the auditory system, Rubel a5 notes that relatively little is known about the maturation of most of the 'integrative' processes that occur in the central regions of the adult auditory pathway. One such process is related to frequency selectivity. Frequency selectivity in adults is studied most often by obtaining a frequency threshold curve (FTC) from a single cell or fiber. This 'tuning' curve is an iso-response contour that describes the stimulus level, at a variety of frequencies, that elicits a criterion level of neuronal activity. The development of FTC's has been reported for inferior colliculus cells in the kitten1, a0, neonatal mouse 45, and bat 5. Another index of frequency selectivity is found in response area curves, and the maturation of this measure has recently been reported for cells in the anterior ventral cochlear nucleus of the kitten 6. The results from these investigations generally reveal that both selectivity and sensitivity are poor in very young animals. The present report describes the maturation
70 of cochlear nucleus evoked response tuning curves in C57BL/6J mice aged between 12 and 65 days. METHODS
Subjects and preparation Mice of the C57BL/6J strain were used throughout since we are currently working with this strain in our laboratory zs. Animals were either bred in the animal quarters of the University of Pennsylvania or were obtained as litters from the Jackson laboratories. The results in subjects from either source were the same. Each mouse was anesthetized with a 20 % urethane solution (1.5 mg/g) using an intraperitoneal injection ag. A tracheotomy was performed and the animal was mounted in a head holder and placed in a sound attenuated chamber. A thermostatically controlled heating pad maintained body temperature at 37 °C. The left external meatus was excised and the calvarium over the left cerebellum was removed. In the youngest neonates the terminal zone of the external auditory meatus 24 was checked and any fluid found there was removed. A stainless steel concentric electrode was lowered into the cochlear nucleus (CN). The center shaft of this electrode was 0.1 m m in diameter and was exposed for 0.25 m m at the tip. The outer sleeve was 0.25 mm in diameter and was also exposed for 0.25 m m at its tip. The vertical separation between the inner and outer contacts was 0.5 m m (the electrodes were manufactured by Rhodes Medical Instruments CO., model SNE-100). The ear was stimulated with 10.0 kHz tone bursts at an intensity of 100 dB, and the activity at the electrode tip was monitored as it approached the CN. The latency, amplitude, and waveshape of the response varied slightly at each age, but with experience and feedback from histology these response parameters could be used to aid in positioning the electrode.
Procedure Eleven groups were formed at 12, 13, 14, 15, 18, 21, 24, 27, 35, 45 and 65 days of age. Five animals were tested on day 12 while 8-10 animals were tested in the remaining groups. After each mouse was prepared a closed-tube sound delivery system was sealed over the exposed tympanic ring 39. The sound pressure level (SPL) of all the pure-tone stimuli were expressed in dB relative to 20 ~Pa. Two stimuli were used; a probe tone of fixed intensity and frequency, and a masker tone of varying frequency and intensity. The probe tone was a 30 msec tone burst (5 msec rise/decay time) presented at a rate of 6/sec. Eight probe-tone frequencies were used: 1.0, 2.5, 5.0, 10.0, 16.0, 20.0, 26.0, and 39.0 kHz. When the masker frequency was present it was a continuous tone. The C N activity was amplified (10,000 ×, bandpass 8-1000 Hz) and displayed on an oscilloscope. 'True' signal averaging was used to enhance the detectability of the tone-burst evoked response and a maximum of 64 individual responses constituted a sample. In many cases the presence of a response could be quickly ascertained and so the sample was less than 64. Two measures of CN activity were sought. The first was the threshold in the quiet for each of the 8 probe-tone frequencies (PFs). At each PF the stimulus was set to
71 between 80 and 90 dB SPL. If an averaged response was observed the level was then attenuated 10 dB and another average obtained. The stimulus was progressively attenuated in 10 dB steps until no response at a visual detection level (VDL) criterion could be recognized. The SPL was then increased by 5 dB and another average obtained. The VDL threshold was defined as the SPL halfway between the lowest intensity at which a response could and could not be detected. A simultaneous masking procedure was then used to obtain tuning curves at six of the probe frequencies. The probe tone was set at a 15 dB sensation level (i.e. 15 dB above the threshold) and this typically evoked a small (5-15 #V, peak-to-peak) response. The masker tone was then introduced at a very low SPL (--10 to --15 dB). If the averaged probe-tone evoked response was still detected, then the masker SPL was increased by 10 dB and another average obtained. This procedure continued with the masker SPL increasing in 10 dB steps until the probe-tone evoked response fell below the VDL. The masker was then attenuated 5 dB and a final average obtained. The masker SPL at the VDL threshold of the probe-tone evoked response was calculated as halfway between the highest masker intensity at which a probe response could and could not be just detected. This procedure was repeated at between 7 and 10 masker frequencies about each of the PFs. One masker frequency was always the same as the PF. The tuning curve thus formed represented the masker SPL (at each of the various masker frequencies) at the VDL threshold of the probe-tone evoked response. In a control experiment a number of mice at 24 days of age had tuning curves measured for the 5.0, 10.0, and 16.0 kHz PF. Tuning curves, however, were obtained for probe-tone sensation levels of 15, 25, 35, 45, and 55 dB. In 26 ~ of the mice (a sample drawn from all age groups) a 40/~A current was passed down the electrode for 20 sec. The animal was decapitated and the head placed in 10 ~ formalin which contained potassium ferrocyanide. The prussian blue reaction was used to verify the electrode location. Since the electrode tip was relatively large and the mouse CN rather small, it was not possible to specify which of the three divisions of the CN the electrode was exactly located in. In all the mice sampled the electrode was located within the borders of the CN and since the amplitudes, latencies, and wave shapes of the remaining animals were similar to those in which the electrode location was verified, we have reason to believe that these electrodes were also in the CN. RESULTS
Thresholds in the quiet From 12 to 24 days threshold sensitivity at all frequencies improved considerably. In general, the mid to high frequencies showed the greatest increase in sensitivity, with the low frequencies exhibiting the least improvement. Beginning around 21 days and extending through 65 days there was a remarkable loss in high frequency threshold sensitivity. At 39.0 kHz, for example, the thresholds went from 36 dB SPL at 21 days to 80 dB SPL at 65 days, a loss of 44 dB. The threshold sensitivity observed between 21 and 27 days was nearly identical to that previously reported for mice 3.
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Fig. 1. The upper panel indicates thresholds in the quiet and evoked response tuning curves measured in 13-day-old mice. The same is shown in the lower panel for 24-day-old mice. The arrows indicate the probe frequency for each of the tuning curves and the vertical bars are i 1 standard deviation. The variability shown is typical of that at the other tuning curves.
Tuning c~lr~es Tuning curves, at all 6 PFs could be obtained for the first time in the 13 day old mice and these appear in the top portion of Fig. 1 along with the thresholds in the quiet. The vertical bars indicate =]= 1 standard deviation for the 10 subjects. This level o f variability was typical of that seen in the other data points. In the bottom portion of Fig. 1 can be seen the data obtained at 24 days of age. The quiet thresholds and the tuning curves are clearly different from the 13-day-old group. The variability remained low (vertical bars) and that shown was typical. The changing shape of the tuning curve with increasing neonatal age is shown in Fig. 2 for 3 PFs and 6 age groups between 12 and 35 days. The tuning curves at 12 days were the most broadly tuned, but even at this early age there was a clear indication of frequency selectivity. For example, a definite trough can be seen around the PF in the 16.0 and 20.0 kHz tuning curve. By 24 days all the tuning curves appeared generally adult-like. A further examination of the data in Fig. 2 reveals that the greatest amount of threshold improvement occurred at the PF, while the least amount of threshold change occurred at the highest and lowest frequency limits of the tuning curve. However, when tuning curve development at both the tip and skirt of the curve was considered in relative developmental terms (i.e. as a percentage of the 35 day response), the rate of threshold change was nearly identical. Since these results show only magnitude differences and not rate of development differences, we have concluded that there was probably a single process contributing to the overall development of the tuning curve.
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Tuning curve Q We have further quantified the tuning curves by calculating a quality (Q) ratio. This ratio represents the center frequency of the curve divided by the bandwidth 10 dB above the center frequency, and provides an indication of the frequency selectivity of the tuning curve. The higher the value of Q, the more sharply tuned (or frequency selective) is the curve. Moreover, since Q is a ratio, comparisons in the sharpness of tuning can be made between curves measured at different PFs and at different ages. The change in Q for the 5.0, 10.0 and 20.0 kHz tuning curves, plotted for each of the 11 age groups, appears in Fig. 3. The change in threshold sensitivity is also shown. These |
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Fig. 3. The development of tuning curve Q and threshold sensitivity (plotted as dB relative to 20/~Pa) is shown for 3 probe frequencies. The vertical bars on the 16.0 kHz Q curve indicate 4- l standard deviation. This level of variability was typical.
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Fig. 5. Tuning curves at two probe frequencies and at multiple sensation levels are shown. Each point is the average of three 24-day-old mice. The insert plots the threshold shift at the characteristic frequency against the probe frequency sensation level. The line of best fit is shown along with the slope, intercept and correlation of the data. Note the similarity of the Q values within each tuning curve at the various sensation levels.
75 that plateau at 6.7 on the 21st day, while in the high frequencies, Q reaches a maximum of 8.9 on the 27th day. The Q of the high frequency tuning curves then declined to a value of 5.6 by the 65th day.
Probe tone level
All the tuning curve data thus far described were obtained at a probe-tone sensation level of 15 dB. Since the thresholds measured in the quiet varied with age, it follows that the tuning curves in the youngest subjects were obtained at higher sound levels than in the older subjects. This raises the possibility that Q varied with stinmlus level rather than age. The data in Fig. 5 show two tuning curves at 10.0 and 16.0 kHz for 5 and 4 probe tone levels respectively. The curves represent the mean data of 3 mice at 24 days of age. The Q values in both curves showed no change over all probe levels. We conclude from these data that the value of Q remains relatively constant, at least over a 45-55 dB change in probe level. Thus, the more broadly tuned curves observed in the youngest groups were not due to the higher SPLs employed. DISCUSSION E v o k e d response tuning curves
The evoked response tuning curve provides an indication of frequency selectivity in much the same way as tuning curves obtained from single auditory nerve fibers s,16,41. This conclusion is based on a number of facts and assumptions, but primarily on the observation that both tuning curves have similar shapes. This is not surprising since both are iso-response contours and represent, in part, the activity of a small segment of the organ of Corti. An important difference, however, is that the fiber tuning curve uses a single tone, whereas the masked tuning curve procedure uses two tones. Two-tone or lateral suppression a7 occurs when the ear is simultaneously stimulated by multiple frequencies. This phenomenon is non-linear and causes a broadening of the tuning curve, as measured both behaviorally and neurophysiologically19,29, a7,44. Thus, the curves in this paper probably indicate the boundaries of the 'suppression area' rather than the 'excitatory area' of the auditory filters. The evoked response tuning curve is a relatively new procedure and there are some points concerning its utility that ought to be mentioned. The experimenter is free to choose his parameters and once these are set, the same measurements can be made from animal to animal. Moreover, the evoked response tuning curves represent a level of neural integration which is greater than that seen for the single cell, yet far below that which contributes to a psychophysically determined tuning curve. It is also our impression that the procedure is robust with respect to electrode placement. Threshold curves and tuning curves always had the same general shape in all the animals within a group. We conclude from this observation that the field potentials evoked by the probe tone are homogenously distributed throughout the CN. Whether this phenomenon on is peculiar to the mouse, or results from the specific recording geometry of the concentric electrode, is not known.
76
Tuning curve development The present results are consistent with the findings of others that frequency selectivity is poor at the inception of hearing !,5,13,3°,45. Tuning curves recorded from kitten inferior colliculus cells have been shown to become more frequency selective between 11 and 40 days 3°. The bandwidth of the internal auditory filters has also been indirectly estimated by behavioral measures of masked threshold sensitivity in neonatal mice 13. The results of this experiment suggest two processes, one occurring between 10 and 12 days and related to inner ear maturation, and the other taking place from 14 to 18 days and related to CNS maturation. During both of these periods frequency selectivity was seen to improve. In the present data during the period of development (i.e. from, 12-24 days) we did not see anything to suggest multiple processes in the maturation of either frequency sensitivity or selectivity. What then governs the development of tunning curves? Frequency selectivity in the form of tuning curves is first observed in events that occur within the cochlea 15,'3~, do. Whether these events are determined by the mechanics of the cochlea alone, or are the end product of mechanical and neural interactions, is a matter of considerable debate 9,15. The basilar membrane, however, is the crucial element in determining the mechanical properties of frequency selectivity", but light and electron microscopy of the mouse cochlea indicate that this structure appears mature between l0 and 14 days of age 21,26. It may be that the elastic properties of the basilar membrane continue to develop beyond the time that this structure appears microscopically mature. The biochemical 4 and electrophysiologica125,2~,4~ environment necessary for normal transduction is also established in the cochlea (day 14-15) before the process of frequency selectivity is mature. Tuning curve development may be influenced by the maturation of the CN and auditory nerve 20. However, the spiral ganglion cells and eighth nerve fibers in the mouse have a normal 'size and configuration' by the 10th day 26, and all subdivisions of the CN exhibit an adult cell population by the 16th day 2s. Efferent fibers have been found in the mouse cochlea very early in development 21 and stimulation of these fibers exerts a strong influence on afferent responses in neonatal kittensL The efferent connections, however, could not have influenced the present results since all testing was conducted on anesthetized animals. Finally, there may be something unique about the C57BL/6J strain and comparisons with other mouse strains (CBA-J, Housemouse, Albino, RB, etc.), on which the bulk of the developmental data have been obtained, may not be appropriate. There is, however, no evidence to suggest that the development of the C57BL/6J cochlea is any different from that reported in other mice 25,39. In adult animals the degree of selectivity in tuning appears to be a property of the inner hair cells 9A°,17. The mouse has between 800 and 950 inner hair cells 14,32 and if the innervation pattern is similar to that found in other mammalian species 3~,43, then between 10 and 20 afferent nerve fibers will synapse on each of these. It follows that the probe-tone evoked activity presently recorded originates primarily from inner hair cells ~2. The development of hair cells has been described for the cat and the hamster2a,83, ~4 and the general pattern appears to be the same in both species. Inner hair cells mature more rapidly than outer hair cells. Furthermore, efferent fibers make
77 the first synaptic contact with inner hair cells. The more numerous afferent fibers then appear to compete with the efferent synapses and eventually supplant them on the soma of the inner hair cella4. The displaced efferent synapses come to lie on the unmyelinated dendrite of the afferent projection to the inner hair cell 4a. Thus, the process of synaptogenesis at the inner hair cell is complex and may well extend beyond the time at which the structures of the organ of Corti appear mature. In summary, we feel that the development of synaptic contacts with the inner hair cells by the afferent dendrites of the auditory nerve, most likely account for the reported development of frequency selectivity. This conclusion must be tempered by the fact that the ontogeny of the 'fine structure' of the basilar membrane, auditory nerve or CN, which may be related to frequency selectivity, is not known. Tuning curve deterioration
Finally, age related losses in auditory function have been reported for C57 mouse strainslS,25,27, 39 and these are associated with hair cell damage11,12,22, 25 and possible degeneration of neurons in the auditory CNS 19. Current evidence supports the hypothesis that inner hair cells are responsible for coding selectivity, while outer hair cells in some way control auditory sensitivityg,10,1L The loss of sensitivity and selectivity at 20.0 and 26.0 kHz, in the 65-day-old mice, indicates degeneration of both outer and inner hair cells in the basal cochlear turn. At I0.0 and 16.0 kHz there was only a loss in threshold sensitivity, and the damage in this region of the cochlea was probably associated with a lesion of the outer hair cells. Thus, the degenerative process in the cochlea of C57BL/6J mice appears to begin in the basal turn (as suggested by Mikaelian and co-worker25, 27) and progresses toward the apex in such a way that outer hair cell loss precedes inner hair cell loss. ACKNOWLEDGEMENTS This work was supported by a grant from the Deafness Research Foundation. The authors gratefully recognize the many fruitful discussions concerning this manuscript with Drs. E. M. Relkin and J. J. Rosowski. We also appreciate the assistance of Ms. Linda Mogul and Mr. Ira Horowitz. K.G.D. is presently a predoctoral student in the Department of Psychology. REFERENCES 1 Aitkin, L. M. and Moore, D. R., inferior colliculus. I1. Development of tuning characteristics and tonotopic organization in central nucleus of the neonatal cat, J. NeurophysioL, 38 (1975) 1208-1216. 2 B6k6sy, G. v., Experiments in hearing, McGraw-Hill, New York, 1960. 3 Bock, G. R. and Saunders, J. C., Effects of low and high-frequency noise bands in producing a physiological correlate of loudness recruitment in mice, Trans. Amer. Acad. OphthaL OtolaryngoL, 82 (1976) 338-342. 4 Bosher, S. K., Morphological and functional changes in the cochlea associated with the inception of hearing, Syrup. zooL Soc. Lond., 37 (1975) 11-22. 5 Brown, P. E., Grinnell, A. D. and Harrison, J. B., The development of hearing in the pallid bat, Antrozous pallidus, J. comp. Physiol., 126 (1978) 169-182.
78 6 Brugge, J. H., Javel, E. and Kitzes, L. M., Signs of functional maturation of peripheral auditory system in discharge patterns of neurons in anteroventral cochlear nucleus of kitten, J. Ne,rvphysiol., 41 (1978) 1557-1579. 7 Carlier, E. and Pujol, R., Early effects of efferent stimulation on the kitten cochlea, Net, rosci. Lett., 3 (1976) 21-27. 8 Dallos, P. and Cheatham, M. A., Compound action potential (AP) tuning curves, J. Acoust. Sot'. Amer., 59 (1976) 591-597. 9 Dallos, P. and Harris, D., Properties of auditory nerve responses in absence of outer hair cells, J. Neurophysiol., 41 (1978) 365-383. 10 Dallos, P., Ryan, A., Harris, D., McGee, T. and Ozdamar, O., Cochlear frequency selectivity in the presence of hair cell damage, In E. F., Evans and J. P. Wilson (Eds.), Psychophysics and Physiology of Hearing, Academic, London, 1977, pp. 249-258. II Deol, M. S., Inherited diseases of the inner ear in man in the light of studies on the mouse, J. reed. Genet., 5 (1968) 137 158. 12 Deol, M. S. and Gluecksohn-Waelsch, S., The role of inner hair cells in hearing, Nature, (Lond.), 278 (1979) 250-252. 13 Ehret, G., Postnatal development in the acoustic system of the housemouse in the light of developing masked thresholds, J. Acoust. Soc. Amer., 62 (1977) 143-148. 14 Ehret, G. and Frankenreiter, M., Quantitative analysis of cochlear structures in the house mouse in relation to mechanisms of acoustic information processing, J. comp. Physiol., 122 (1977) 65-85. 15 Evans, E. F., Cochlear nerve and cochlear nucleus. In W. D. Keidel and W. D. Neff (Eds.), Handbook of Sensory Physiology, Vol. 2, Auditory System: Physiology (CNS), Behavioral Studies, Psychoacoustics, Springer-Verlag, New York, 1975, pp. 1-108. 16 Finck, A., Physiological correlate of tonal masking, J. Acoust. Soc. Amer., 39 (1966) 1056-1062. 17 Harris, D. M., Action potential suppression, tuning curves and thresholds: Comparison with single fiber data, Hearing Res., 1 (1979) 133-154. 18 Henry, K. R. and Lepkowski, C. M., Evoked potential correlates of genetic progressive hearing loss, Aeta oto-laryng., 1,366-374. 19 Houtgast, T., Psychophysical experiments on 'tuning curves' and 'two-tone inhibition', Aeoustica, 29 (1973) 168-179. 20 Kane, E. S. and Habib, C. P., Development of the dorsal cochlear nucleus of the cat: an electron microscopic study, Amer. J. Anat., 153 (1978) 321-343. 21 Kikuchi, K. and Hilding, D., The development of the organ of Corti in the mouse, Acta otolaryng., 60 (1965) 20%221. 22 Kocher, W, Untersuchungen zur Genetik und Pathologic der Entwicklung sp~t einsetzender heredit/irer Taubheit bei der Maus (Mus musculus), Arch. Ohr.-, Nas.- u. Kehlk.-Heilk., 177 (1960) 108-145. 23 Lindemann, H. H., Ades, H. W., Bredberg, G. and Engstr6m, H., The sensory hairs and the rectorial membrane in the development of the cat's organ of Corti, Acta oto-laryng., 72 (1971) 229-242. 24 Maio, F. H. and Tonndorf, J., The terminal zone of the external auditory meatus in a variety of mammals, Arch. Otolaryng., 104 (1978) 570-575. 25 Mikaelian, D. O., Development and degeneration of hearing in the C57/B16 mouse: relation of electrophysiologic responses from round window and cochlear nucleus to cochlear anatomy and behavioral responses, Laryngoscope (St. Louis), 89 (1979) 1-15. 26 Mikaelian, D. and Ruben, R. J., Development of hearing in the normal CBA-J mouse, Acta oto-laryng., 59 (1965) 451~461. 27 Mikaelian, D. O., Warfield, D. and Norris, O., Genetic progressive hearing loss in the C57/B16 mouse, Acta oto-laryng., 77 (1974) 327-334. 28 Milonyeni, M., The late stages of the development of the primary cochlear nuclei in mice, Brain Research, 4 (1967) 334-344. 29 Moore, B. C., Psychophysical tuning curves measured in simultaneous and foward masking, J. Aeoust. Soe. Amer., 60 (1978) 524-532. 30 Moore, D. R. and lrvine, D. R. F., The development of some peripheral and central auditory responses in the neonatal cat, Brain Research, 163 (1979) 49-59. 31 Morrison, D., Schindler, R. A. and Wers/ill, J., A quantitative analysis of the afferent innervation of the organ of Corti in the guinea pig, Aeta oto-laryng., 79 (1975) 11-23.
79 32 Norris, C. H., Cawthon, T. H. and Carroll, R. C., Kanamycin priming for audiogenic seizures in mice, Neuropharmacology, 16 (1977) 375-380. 33 Pujol. R. and Abonnenc, M., Receptor maturation and synaptogenesis in the golden hamster cochlea, Arch. oto-rhino-laryng., 217 (1977) 1-12. 34 Pujol, R., Carlier, E. and Devigne, C., Different patterns of cochlear innervation during the development of the kitten, J. comp. Neurol., 177 (1978) 529-535. 35 Rubel, E. W., Ontogeny of structure and function in the vertebrate auditory system. In M. Jacobson (Ed.), Handbook of Sensory Physiology, 11"ol. IX. Development of Sensory Systems, Springer-Verlag,New York, 1978, pp. 135-237. 36 Russell, I. J. and Sellick, P. M., Intracellular studies of hair cells in the mammalian cochlea, J. Physiol. (Lond.), 284 (1978) 261-290. 37 Sachs, M. B. and Kiang, N. Y.-S., Two tone inhibition in auditory nerve fibers, J. Acoust. Soc. Amer., 43 (1968) 1120-1128. 38 Saunders. J. C. and Bock, G. R., Influences of early auditory trauma on auditory development, In G. Gottlieb (Ed.), Studies on the Development of Behavior and the Nervous System, 1Iol. 4, Early influences, Academic, New York, 1978, pp. 249-287. 39 Saunders, J. C. and Hirsch, K. A., Changes in cochlear microphonic sensitivity after priming C57BL/6J mice at various ages for audiogenic seizures, J. comp. Physiol. Psychol., 90 (1976) 212-220. 40 Saunders, J. C. and Rosowski, J. J., Assessment of hearing in animals. In W, F. Rintelmann (Ed.), The Assessment of Hearing, University Press, Baltimore, 1979, pp. 487-529. 41 Saunders, J. C., Rosowski, J. J., and Pallone, R. L., Frequency selectivity in parakeet hearing: behavioral and physiological evidence. In Proc. 17th Int. Congr. Ornithology, Springer-Verlag, Berlin, 1979, in press. 42 Schmidt, R. S. and Fernandez, C., Development of mammalian endocochlear potential, J. exp. Zool., 153 (1963) 227-236. 43 Spoendlin, H.,The innervation of the cochlear receptor, In A. Moller (Ed.), Basic Mechanisms in Hearing, Academic, New York, 1973, p. 185-230. 44 Vogten, L. L. M., Low-level pure-tone masking: a comparison of 'tuning curves' obtained with simultaneous and foward masking, J. Acoust. Soc. Amer., 63 (1978) 1520-1527. 45 Willott, J. F.,and Shnerson, A., Rapid development of tuning characteristics of inferior colliculus neurons of mouse pups, Brain Research, 148 (1978) 230-233.
69
T H E M A T U R A T I O N OF F R E Q U E N C Y SELECTIVITY IN C57BL/6J MICE S T U D I E D W I T H A U D I T O R Y EVOKED RESPONSE T U N I N G CURVES
JAMES C. SAUNDERS, KIM G. DOLGIN and LOUIS D. LOWRY Department of Otorhinolaryngology and Human Communication and Department of Psychology, University of Pennsylvania, Philadelphia, Pa, 19104 (U.S.A.)
(Accepted August 30th, 1979) Key words: frequency selectivity - - tuning curves - - auditory development - - cochlear nucleus
SUMMARY The present study was designed to examine the ontogeny of frequency selectivity in the neonatal auditory system. Mice were tested between 12 and 65 days of age. At each age two measures of auditory sensitivity were made from cochlear nucleus evoked responses. Tone-burst evoked-response thresholds in the quiet were determined for frequencies between 1.0 and 39.0 kHz. A two-tone simultaneous masking procedure was then used to obtain evoked response tuning curves. The frequency selectivity of the tuning curves was quantified by calculating a Q ratio. The results show that tuning is poor in neonates but rapidly improves to adult-like levels within 5-16 days after the inception of auditory function. The data also indicate that the development of frequency selectivity varies directly with the maturation of threshold sensitivity.
INTRODUCTION In his recent review of structural and functional developments within the auditory system, Rubel a5 notes that relatively little is known about the maturation of most of the 'integrative' processes that occur in the central regions of the adult auditory pathway. One such process is related to frequency selectivity. Frequency selectivity in adults is studied most often by obtaining a frequency threshold curve (FTC) from a single cell or fiber. This 'tuning' curve is an iso-response contour that describes the stimulus level, at a variety of frequencies, that elicits a criterion level of neuronal activity. The development of FTC's has been reported for inferior colliculus cells in the kitten1, a0, neonatal mouse 45, and bat 5. Another index of frequency selectivity is found in response area curves, and the maturation of this measure has recently been reported for cells in the anterior ventral cochlear nucleus of the kitten 6. The results from these investigations generally reveal that both selectivity and sensitivity are poor in very young animals. The present report describes the maturation
70 of cochlear nucleus evoked response tuning curves in C57BL/6J mice aged between 12 and 65 days. METHODS
Subjects and preparation Mice of the C57BL/6J strain were used throughout since we are currently working with this strain in our laboratory zs. Animals were either bred in the animal quarters of the University of Pennsylvania or were obtained as litters from the Jackson laboratories. The results in subjects from either source were the same. Each mouse was anesthetized with a 20 % urethane solution (1.5 mg/g) using an intraperitoneal injection ag. A tracheotomy was performed and the animal was mounted in a head holder and placed in a sound attenuated chamber. A thermostatically controlled heating pad maintained body temperature at 37 °C. The left external meatus was excised and the calvarium over the left cerebellum was removed. In the youngest neonates the terminal zone of the external auditory meatus 24 was checked and any fluid found there was removed. A stainless steel concentric electrode was lowered into the cochlear nucleus (CN). The center shaft of this electrode was 0.1 m m in diameter and was exposed for 0.25 m m at the tip. The outer sleeve was 0.25 mm in diameter and was also exposed for 0.25 m m at its tip. The vertical separation between the inner and outer contacts was 0.5 m m (the electrodes were manufactured by Rhodes Medical Instruments CO., model SNE-100). The ear was stimulated with 10.0 kHz tone bursts at an intensity of 100 dB, and the activity at the electrode tip was monitored as it approached the CN. The latency, amplitude, and waveshape of the response varied slightly at each age, but with experience and feedback from histology these response parameters could be used to aid in positioning the electrode.
Procedure Eleven groups were formed at 12, 13, 14, 15, 18, 21, 24, 27, 35, 45 and 65 days of age. Five animals were tested on day 12 while 8-10 animals were tested in the remaining groups. After each mouse was prepared a closed-tube sound delivery system was sealed over the exposed tympanic ring 39. The sound pressure level (SPL) of all the pure-tone stimuli were expressed in dB relative to 20 ~Pa. Two stimuli were used; a probe tone of fixed intensity and frequency, and a masker tone of varying frequency and intensity. The probe tone was a 30 msec tone burst (5 msec rise/decay time) presented at a rate of 6/sec. Eight probe-tone frequencies were used: 1.0, 2.5, 5.0, 10.0, 16.0, 20.0, 26.0, and 39.0 kHz. When the masker frequency was present it was a continuous tone. The C N activity was amplified (10,000 ×, bandpass 8-1000 Hz) and displayed on an oscilloscope. 'True' signal averaging was used to enhance the detectability of the tone-burst evoked response and a maximum of 64 individual responses constituted a sample. In many cases the presence of a response could be quickly ascertained and so the sample was less than 64. Two measures of CN activity were sought. The first was the threshold in the quiet for each of the 8 probe-tone frequencies (PFs). At each PF the stimulus was set to
71 between 80 and 90 dB SPL. If an averaged response was observed the level was then attenuated 10 dB and another average obtained. The stimulus was progressively attenuated in 10 dB steps until no response at a visual detection level (VDL) criterion could be recognized. The SPL was then increased by 5 dB and another average obtained. The VDL threshold was defined as the SPL halfway between the lowest intensity at which a response could and could not be detected. A simultaneous masking procedure was then used to obtain tuning curves at six of the probe frequencies. The probe tone was set at a 15 dB sensation level (i.e. 15 dB above the threshold) and this typically evoked a small (5-15 #V, peak-to-peak) response. The masker tone was then introduced at a very low SPL (--10 to --15 dB). If the averaged probe-tone evoked response was still detected, then the masker SPL was increased by 10 dB and another average obtained. This procedure continued with the masker SPL increasing in 10 dB steps until the probe-tone evoked response fell below the VDL. The masker was then attenuated 5 dB and a final average obtained. The masker SPL at the VDL threshold of the probe-tone evoked response was calculated as halfway between the highest masker intensity at which a probe response could and could not be just detected. This procedure was repeated at between 7 and 10 masker frequencies about each of the PFs. One masker frequency was always the same as the PF. The tuning curve thus formed represented the masker SPL (at each of the various masker frequencies) at the VDL threshold of the probe-tone evoked response. In a control experiment a number of mice at 24 days of age had tuning curves measured for the 5.0, 10.0, and 16.0 kHz PF. Tuning curves, however, were obtained for probe-tone sensation levels of 15, 25, 35, 45, and 55 dB. In 26 ~ of the mice (a sample drawn from all age groups) a 40/~A current was passed down the electrode for 20 sec. The animal was decapitated and the head placed in 10 ~ formalin which contained potassium ferrocyanide. The prussian blue reaction was used to verify the electrode location. Since the electrode tip was relatively large and the mouse CN rather small, it was not possible to specify which of the three divisions of the CN the electrode was exactly located in. In all the mice sampled the electrode was located within the borders of the CN and since the amplitudes, latencies, and wave shapes of the remaining animals were similar to those in which the electrode location was verified, we have reason to believe that these electrodes were also in the CN. RESULTS
Thresholds in the quiet From 12 to 24 days threshold sensitivity at all frequencies improved considerably. In general, the mid to high frequencies showed the greatest increase in sensitivity, with the low frequencies exhibiting the least improvement. Beginning around 21 days and extending through 65 days there was a remarkable loss in high frequency threshold sensitivity. At 39.0 kHz, for example, the thresholds went from 36 dB SPL at 21 days to 80 dB SPL at 65 days, a loss of 44 dB. The threshold sensitivity observed between 21 and 27 days was nearly identical to that previously reported for mice 3.
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Fig. 1. The upper panel indicates thresholds in the quiet and evoked response tuning curves measured in 13-day-old mice. The same is shown in the lower panel for 24-day-old mice. The arrows indicate the probe frequency for each of the tuning curves and the vertical bars are i 1 standard deviation. The variability shown is typical of that at the other tuning curves.
Tuning c~lr~es Tuning curves, at all 6 PFs could be obtained for the first time in the 13 day old mice and these appear in the top portion of Fig. 1 along with the thresholds in the quiet. The vertical bars indicate =]= 1 standard deviation for the 10 subjects. This level o f variability was typical of that seen in the other data points. In the bottom portion of Fig. 1 can be seen the data obtained at 24 days of age. The quiet thresholds and the tuning curves are clearly different from the 13-day-old group. The variability remained low (vertical bars) and that shown was typical. The changing shape of the tuning curve with increasing neonatal age is shown in Fig. 2 for 3 PFs and 6 age groups between 12 and 35 days. The tuning curves at 12 days were the most broadly tuned, but even at this early age there was a clear indication of frequency selectivity. For example, a definite trough can be seen around the PF in the 16.0 and 20.0 kHz tuning curve. By 24 days all the tuning curves appeared generally adult-like. A further examination of the data in Fig. 2 reveals that the greatest amount of threshold improvement occurred at the PF, while the least amount of threshold change occurred at the highest and lowest frequency limits of the tuning curve. However, when tuning curve development at both the tip and skirt of the curve was considered in relative developmental terms (i.e. as a percentage of the 35 day response), the rate of threshold change was nearly identical. Since these results show only magnitude differences and not rate of development differences, we have concluded that there was probably a single process contributing to the overall development of the tuning curve.
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Tuning curve Q We have further quantified the tuning curves by calculating a quality (Q) ratio. This ratio represents the center frequency of the curve divided by the bandwidth 10 dB above the center frequency, and provides an indication of the frequency selectivity of the tuning curve. The higher the value of Q, the more sharply tuned (or frequency selective) is the curve. Moreover, since Q is a ratio, comparisons in the sharpness of tuning can be made between curves measured at different PFs and at different ages. The change in Q for the 5.0, 10.0 and 20.0 kHz tuning curves, plotted for each of the 11 age groups, appears in Fig. 3. The change in threshold sensitivity is also shown. These |
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Fig. 5. Tuning curves at two probe frequencies and at multiple sensation levels are shown. Each point is the average of three 24-day-old mice. The insert plots the threshold shift at the characteristic frequency against the probe frequency sensation level. The line of best fit is shown along with the slope, intercept and correlation of the data. Note the similarity of the Q values within each tuning curve at the various sensation levels.
75 that plateau at 6.7 on the 21st day, while in the high frequencies, Q reaches a maximum of 8.9 on the 27th day. The Q of the high frequency tuning curves then declined to a value of 5.6 by the 65th day.
Probe tone level
All the tuning curve data thus far described were obtained at a probe-tone sensation level of 15 dB. Since the thresholds measured in the quiet varied with age, it follows that the tuning curves in the youngest subjects were obtained at higher sound levels than in the older subjects. This raises the possibility that Q varied with stinmlus level rather than age. The data in Fig. 5 show two tuning curves at 10.0 and 16.0 kHz for 5 and 4 probe tone levels respectively. The curves represent the mean data of 3 mice at 24 days of age. The Q values in both curves showed no change over all probe levels. We conclude from these data that the value of Q remains relatively constant, at least over a 45-55 dB change in probe level. Thus, the more broadly tuned curves observed in the youngest groups were not due to the higher SPLs employed. DISCUSSION E v o k e d response tuning curves
The evoked response tuning curve provides an indication of frequency selectivity in much the same way as tuning curves obtained from single auditory nerve fibers s,16,41. This conclusion is based on a number of facts and assumptions, but primarily on the observation that both tuning curves have similar shapes. This is not surprising since both are iso-response contours and represent, in part, the activity of a small segment of the organ of Corti. An important difference, however, is that the fiber tuning curve uses a single tone, whereas the masked tuning curve procedure uses two tones. Two-tone or lateral suppression a7 occurs when the ear is simultaneously stimulated by multiple frequencies. This phenomenon is non-linear and causes a broadening of the tuning curve, as measured both behaviorally and neurophysiologically19,29, a7,44. Thus, the curves in this paper probably indicate the boundaries of the 'suppression area' rather than the 'excitatory area' of the auditory filters. The evoked response tuning curve is a relatively new procedure and there are some points concerning its utility that ought to be mentioned. The experimenter is free to choose his parameters and once these are set, the same measurements can be made from animal to animal. Moreover, the evoked response tuning curves represent a level of neural integration which is greater than that seen for the single cell, yet far below that which contributes to a psychophysically determined tuning curve. It is also our impression that the procedure is robust with respect to electrode placement. Threshold curves and tuning curves always had the same general shape in all the animals within a group. We conclude from this observation that the field potentials evoked by the probe tone are homogenously distributed throughout the CN. Whether this phenomenon on is peculiar to the mouse, or results from the specific recording geometry of the concentric electrode, is not known.
76
Tuning curve development The present results are consistent with the findings of others that frequency selectivity is poor at the inception of hearing !,5,13,3°,45. Tuning curves recorded from kitten inferior colliculus cells have been shown to become more frequency selective between 11 and 40 days 3°. The bandwidth of the internal auditory filters has also been indirectly estimated by behavioral measures of masked threshold sensitivity in neonatal mice 13. The results of this experiment suggest two processes, one occurring between 10 and 12 days and related to inner ear maturation, and the other taking place from 14 to 18 days and related to CNS maturation. During both of these periods frequency selectivity was seen to improve. In the present data during the period of development (i.e. from, 12-24 days) we did not see anything to suggest multiple processes in the maturation of either frequency sensitivity or selectivity. What then governs the development of tunning curves? Frequency selectivity in the form of tuning curves is first observed in events that occur within the cochlea 15,'3~, do. Whether these events are determined by the mechanics of the cochlea alone, or are the end product of mechanical and neural interactions, is a matter of considerable debate 9,15. The basilar membrane, however, is the crucial element in determining the mechanical properties of frequency selectivity", but light and electron microscopy of the mouse cochlea indicate that this structure appears mature between l0 and 14 days of age 21,26. It may be that the elastic properties of the basilar membrane continue to develop beyond the time that this structure appears microscopically mature. The biochemical 4 and electrophysiologica125,2~,4~ environment necessary for normal transduction is also established in the cochlea (day 14-15) before the process of frequency selectivity is mature. Tuning curve development may be influenced by the maturation of the CN and auditory nerve 20. However, the spiral ganglion cells and eighth nerve fibers in the mouse have a normal 'size and configuration' by the 10th day 26, and all subdivisions of the CN exhibit an adult cell population by the 16th day 2s. Efferent fibers have been found in the mouse cochlea very early in development 21 and stimulation of these fibers exerts a strong influence on afferent responses in neonatal kittensL The efferent connections, however, could not have influenced the present results since all testing was conducted on anesthetized animals. Finally, there may be something unique about the C57BL/6J strain and comparisons with other mouse strains (CBA-J, Housemouse, Albino, RB, etc.), on which the bulk of the developmental data have been obtained, may not be appropriate. There is, however, no evidence to suggest that the development of the C57BL/6J cochlea is any different from that reported in other mice 25,39. In adult animals the degree of selectivity in tuning appears to be a property of the inner hair cells 9A°,17. The mouse has between 800 and 950 inner hair cells 14,32 and if the innervation pattern is similar to that found in other mammalian species 3~,43, then between 10 and 20 afferent nerve fibers will synapse on each of these. It follows that the probe-tone evoked activity presently recorded originates primarily from inner hair cells ~2. The development of hair cells has been described for the cat and the hamster2a,83, ~4 and the general pattern appears to be the same in both species. Inner hair cells mature more rapidly than outer hair cells. Furthermore, efferent fibers make
77 the first synaptic contact with inner hair cells. The more numerous afferent fibers then appear to compete with the efferent synapses and eventually supplant them on the soma of the inner hair cella4. The displaced efferent synapses come to lie on the unmyelinated dendrite of the afferent projection to the inner hair cell 4a. Thus, the process of synaptogenesis at the inner hair cell is complex and may well extend beyond the time at which the structures of the organ of Corti appear mature. In summary, we feel that the development of synaptic contacts with the inner hair cells by the afferent dendrites of the auditory nerve, most likely account for the reported development of frequency selectivity. This conclusion must be tempered by the fact that the ontogeny of the 'fine structure' of the basilar membrane, auditory nerve or CN, which may be related to frequency selectivity, is not known. Tuning curve deterioration
Finally, age related losses in auditory function have been reported for C57 mouse strainslS,25,27, 39 and these are associated with hair cell damage11,12,22, 25 and possible degeneration of neurons in the auditory CNS 19. Current evidence supports the hypothesis that inner hair cells are responsible for coding selectivity, while outer hair cells in some way control auditory sensitivityg,10,1L The loss of sensitivity and selectivity at 20.0 and 26.0 kHz, in the 65-day-old mice, indicates degeneration of both outer and inner hair cells in the basal cochlear turn. At I0.0 and 16.0 kHz there was only a loss in threshold sensitivity, and the damage in this region of the cochlea was probably associated with a lesion of the outer hair cells. Thus, the degenerative process in the cochlea of C57BL/6J mice appears to begin in the basal turn (as suggested by Mikaelian and co-worker25, 27) and progresses toward the apex in such a way that outer hair cell loss precedes inner hair cell loss. ACKNOWLEDGEMENTS This work was supported by a grant from the Deafness Research Foundation. The authors gratefully recognize the many fruitful discussions concerning this manuscript with Drs. E. M. Relkin and J. J. Rosowski. We also appreciate the assistance of Ms. Linda Mogul and Mr. Ira Horowitz. K.G.D. is presently a predoctoral student in the Department of Psychology. REFERENCES 1 Aitkin, L. M. and Moore, D. R., inferior colliculus. I1. Development of tuning characteristics and tonotopic organization in central nucleus of the neonatal cat, J. NeurophysioL, 38 (1975) 1208-1216. 2 B6k6sy, G. v., Experiments in hearing, McGraw-Hill, New York, 1960. 3 Bock, G. R. and Saunders, J. C., Effects of low and high-frequency noise bands in producing a physiological correlate of loudness recruitment in mice, Trans. Amer. Acad. OphthaL OtolaryngoL, 82 (1976) 338-342. 4 Bosher, S. K., Morphological and functional changes in the cochlea associated with the inception of hearing, Syrup. zooL Soc. Lond., 37 (1975) 11-22. 5 Brown, P. E., Grinnell, A. D. and Harrison, J. B., The development of hearing in the pallid bat, Antrozous pallidus, J. comp. Physiol., 126 (1978) 169-182.
78 6 Brugge, J. H., Javel, E. and Kitzes, L. M., Signs of functional maturation of peripheral auditory system in discharge patterns of neurons in anteroventral cochlear nucleus of kitten, J. Ne,rvphysiol., 41 (1978) 1557-1579. 7 Carlier, E. and Pujol, R., Early effects of efferent stimulation on the kitten cochlea, Net, rosci. Lett., 3 (1976) 21-27. 8 Dallos, P. and Cheatham, M. A., Compound action potential (AP) tuning curves, J. Acoust. Sot'. Amer., 59 (1976) 591-597. 9 Dallos, P. and Harris, D., Properties of auditory nerve responses in absence of outer hair cells, J. Neurophysiol., 41 (1978) 365-383. 10 Dallos, P., Ryan, A., Harris, D., McGee, T. and Ozdamar, O., Cochlear frequency selectivity in the presence of hair cell damage, In E. F., Evans and J. P. Wilson (Eds.), Psychophysics and Physiology of Hearing, Academic, London, 1977, pp. 249-258. II Deol, M. S., Inherited diseases of the inner ear in man in the light of studies on the mouse, J. reed. Genet., 5 (1968) 137 158. 12 Deol, M. S. and Gluecksohn-Waelsch, S., The role of inner hair cells in hearing, Nature, (Lond.), 278 (1979) 250-252. 13 Ehret, G., Postnatal development in the acoustic system of the housemouse in the light of developing masked thresholds, J. Acoust. Soc. Amer., 62 (1977) 143-148. 14 Ehret, G. and Frankenreiter, M., Quantitative analysis of cochlear structures in the house mouse in relation to mechanisms of acoustic information processing, J. comp. Physiol., 122 (1977) 65-85. 15 Evans, E. F., Cochlear nerve and cochlear nucleus. In W. D. Keidel and W. D. Neff (Eds.), Handbook of Sensory Physiology, Vol. 2, Auditory System: Physiology (CNS), Behavioral Studies, Psychoacoustics, Springer-Verlag, New York, 1975, pp. 1-108. 16 Finck, A., Physiological correlate of tonal masking, J. Acoust. Soc. Amer., 39 (1966) 1056-1062. 17 Harris, D. M., Action potential suppression, tuning curves and thresholds: Comparison with single fiber data, Hearing Res., 1 (1979) 133-154. 18 Henry, K. R. and Lepkowski, C. M., Evoked potential correlates of genetic progressive hearing loss, Aeta oto-laryng., 1,366-374. 19 Houtgast, T., Psychophysical experiments on 'tuning curves' and 'two-tone inhibition', Aeoustica, 29 (1973) 168-179. 20 Kane, E. S. and Habib, C. P., Development of the dorsal cochlear nucleus of the cat: an electron microscopic study, Amer. J. Anat., 153 (1978) 321-343. 21 Kikuchi, K. and Hilding, D., The development of the organ of Corti in the mouse, Acta otolaryng., 60 (1965) 20%221. 22 Kocher, W, Untersuchungen zur Genetik und Pathologic der Entwicklung sp~t einsetzender heredit/irer Taubheit bei der Maus (Mus musculus), Arch. Ohr.-, Nas.- u. Kehlk.-Heilk., 177 (1960) 108-145. 23 Lindemann, H. H., Ades, H. W., Bredberg, G. and Engstr6m, H., The sensory hairs and the rectorial membrane in the development of the cat's organ of Corti, Acta oto-laryng., 72 (1971) 229-242. 24 Maio, F. H. and Tonndorf, J., The terminal zone of the external auditory meatus in a variety of mammals, Arch. Otolaryng., 104 (1978) 570-575. 25 Mikaelian, D. O., Development and degeneration of hearing in the C57/B16 mouse: relation of electrophysiologic responses from round window and cochlear nucleus to cochlear anatomy and behavioral responses, Laryngoscope (St. Louis), 89 (1979) 1-15. 26 Mikaelian, D. and Ruben, R. J., Development of hearing in the normal CBA-J mouse, Acta oto-laryng., 59 (1965) 451~461. 27 Mikaelian, D. O., Warfield, D. and Norris, O., Genetic progressive hearing loss in the C57/B16 mouse, Acta oto-laryng., 77 (1974) 327-334. 28 Milonyeni, M., The late stages of the development of the primary cochlear nuclei in mice, Brain Research, 4 (1967) 334-344. 29 Moore, B. C., Psychophysical tuning curves measured in simultaneous and foward masking, J. Aeoust. Soe. Amer., 60 (1978) 524-532. 30 Moore, D. R. and lrvine, D. R. F., The development of some peripheral and central auditory responses in the neonatal cat, Brain Research, 163 (1979) 49-59. 31 Morrison, D., Schindler, R. A. and Wers/ill, J., A quantitative analysis of the afferent innervation of the organ of Corti in the guinea pig, Aeta oto-laryng., 79 (1975) 11-23.
79 32 Norris, C. H., Cawthon, T. H. and Carroll, R. C., Kanamycin priming for audiogenic seizures in mice, Neuropharmacology, 16 (1977) 375-380. 33 Pujol. R. and Abonnenc, M., Receptor maturation and synaptogenesis in the golden hamster cochlea, Arch. oto-rhino-laryng., 217 (1977) 1-12. 34 Pujol, R., Carlier, E. and Devigne, C., Different patterns of cochlear innervation during the development of the kitten, J. comp. Neurol., 177 (1978) 529-535. 35 Rubel, E. W., Ontogeny of structure and function in the vertebrate auditory system. In M. Jacobson (Ed.), Handbook of Sensory Physiology, 11"ol. IX. Development of Sensory Systems, Springer-Verlag,New York, 1978, pp. 135-237. 36 Russell, I. J. and Sellick, P. M., Intracellular studies of hair cells in the mammalian cochlea, J. Physiol. (Lond.), 284 (1978) 261-290. 37 Sachs, M. B. and Kiang, N. Y.-S., Two tone inhibition in auditory nerve fibers, J. Acoust. Soc. Amer., 43 (1968) 1120-1128. 38 Saunders. J. C. and Bock, G. R., Influences of early auditory trauma on auditory development, In G. Gottlieb (Ed.), Studies on the Development of Behavior and the Nervous System, 1Iol. 4, Early influences, Academic, New York, 1978, pp. 249-287. 39 Saunders, J. C. and Hirsch, K. A., Changes in cochlear microphonic sensitivity after priming C57BL/6J mice at various ages for audiogenic seizures, J. comp. Physiol. Psychol., 90 (1976) 212-220. 40 Saunders, J. C. and Rosowski, J. J., Assessment of hearing in animals. In W, F. Rintelmann (Ed.), The Assessment of Hearing, University Press, Baltimore, 1979, pp. 487-529. 41 Saunders, J. C., Rosowski, J. J., and Pallone, R. L., Frequency selectivity in parakeet hearing: behavioral and physiological evidence. In Proc. 17th Int. Congr. Ornithology, Springer-Verlag, Berlin, 1979, in press. 42 Schmidt, R. S. and Fernandez, C., Development of mammalian endocochlear potential, J. exp. Zool., 153 (1963) 227-236. 43 Spoendlin, H.,The innervation of the cochlear receptor, In A. Moller (Ed.), Basic Mechanisms in Hearing, Academic, New York, 1973, p. 185-230. 44 Vogten, L. L. M., Low-level pure-tone masking: a comparison of 'tuning curves' obtained with simultaneous and foward masking, J. Acoust. Soc. Amer., 63 (1978) 1520-1527. 45 Willott, J. F.,and Shnerson, A., Rapid development of tuning characteristics of inferior colliculus neurons of mouse pups, Brain Research, 148 (1978) 230-233.