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Combined cochleo-saccular and neuroepithelial abnormalities in the Varitint-waddler-J (VaJ) mouse
Hearing Research 123 (1998) 125^136
Combined cochleo-saccular and neuroepithelial abnormalities in the Varitint-waddler-J (VaJ ) mouse Joanne Cable a
a;b;
*, Karen P. Steel
a
MRC Institute of Hearing Research, University Park, Nottingham, NG7 2RD, UK b School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, UK Received 21 January 1998; revised 18 May 1998; accepted 23 May 1998
Abstract Hearing loss in Varitint-waddler-J (VaJ ) mice is of mixed origin with both cochleo-saccular and neuroepithelial components. Both VaJ /VaJ and VaJ /+ mutants show impaired cochlear function, but the homozygotes are more severely affected than heterozygotes. Neither group have any detectable compound action potential. Cochlear microphonics are only seen in half of the heterozygotes, at a reduced amplitude and raised threshold, and are not detected in any homozygotes. Summating potentials (SP) responses are seen in most of the heterozygotes, at high stimulus levels. The only responses in homozygotes were negative SPs seen in half of the mutants at very high sound levels, while the remaining homozygotes showed no responses to sound stimulation. Endocochlear potentials (EP) were often small or absent in both groups of mutants, with the homozygotes being more severely affected. Reduced pigmentation in the stria vascularis appears to be associated with a reduced EP, while a primary defect of the neuroepithelium, detectable by electron microscopy in hair cells of 14 day old mice, dramatically influences evoked potentials. z 1998 Elsevier Science B.V. All rights reserved. Key words: Endocochlear potential; Stria vascularis; Melanocyte; Compound action potential; Summating potential; Mouse mutant; Pigment defect
1. Introduction Numerous mouse mutants with hearing defects are available as models for understanding human deafness (Steel, 1991, 1995). Functional and anatomical studies of the mouse and human inner ear have identi¢ed the same broad categories of pathology in the two species : morphogenetic, cochleo-saccular and neuroepithelial. Morphogenetic abnormalities involve gross structural deformities of the labyrinth. A strial abnormality is the primary cochleo-saccular defect in which there is a reduced or absent EP and sometimes collapse of Reissner's membrane, which eventually results in degeneration of the organ of Corti and spiral ganglion cells. Neuroepithelial defects originate in the organ of Corti and do not a¡ect the stria directly (Steel and Bock, 1983a). * Corresponding author. School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK. Tel.: +44 (117) 9287473; Fax: +44 (117) 9257374; E-mail: [email protected]
Physiological and structural analyses of all these mouse mutants provides important information on the biological basis of normal hearing and genetic deafness. Unravelling the complexity of the inner ear has progressed more recently to identifying genes and their mutations which a¡ect normal hearing (Steel and Brown, 1994). To date, 41 loci have been identi¢ed that are involved in non-syndromic deafness (Hereditary Hearing Impairment homepage http://dnalab.www.uia.ac.be/dnalab/ hhh/). The full value of data emerging on the genetics of deafness derived from studies of mouse mutants and their human homologues will be maximized if it can be examined in the light of known functional and structural abnormalities of a particular mouse mutant. The Varitint-waddler-J mouse (VaJ ) arose as a semidominant mutation on chromosome 12 from the Va allele (Lane, 1972; Silvers, 1979). The Va mouse was ¢rst observed by Cloudman and Bunker (1945) in an outcross between C57 black and C57 brown stocks. Va/
0378-5955 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 0 7 - 5
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+ mice display a combination of spotting with dilution of coat colour and exhibit near normal viability. The homozygotes are white except for small patches of coloured hairs on the ears and tail base. Va/Va males are sterile and many mice of this genotype die before birth. All Va mice are deaf and show circling behaviour, head tossing and hyperactivity (Cools, 1972a,b). Deol (1954) examining the inner ear morphology of Va/+ mice noted the ¢rst defects 4 days after birth. Anomalies of the tectorial membrane, followed by abnormalities in the spiral ganglion, organ of Corti and then strial atrophy are seen in the Va heterozygotes and homozygotes, characteristic of a neuroepithelial defect. The e¡ects of the VaJ mutation appear to be less severe than those associated with Va. VaJ /+ have only a slightly diluted coat colour (resembling Wv /+ mice). Both VaJ /+ and VaJ /VaJ are deaf but they behave normally and are fertile although a number of homozygotes die in utero (Lane, 1972). This study was undertaken to examine the physiological and structural abnormalities of the inner ear of VaJ mice. 2. Materials and methods Mice carrying the VaJ allele on a C57BL/6/C3H F1 genetic background were obtained from the Jackson Laboratory, and a breeding colony established without the Hd mutation which originally was segregating in the supplied stock. Heterozygous mice were intercrossed to generate VaJ /VaJ , VaJ /+ and +/+ mice, easily distinguished by their coat colours. All control mice had a black coat, pigmented pinna, tail, and feet. Heterozygotes had a grey diluted coat colour and a white belly spot often with an associated black patch. Their feet were white but the tails were at least partially pigmented. Homozygotes had a white dorsal coat with grey £ecks and some black patches. The typical white ventral surface had occasional grey patches. Pigmented patches when present on the head were often asymmetrical. The pinna and tail were pigmented or partially pigmented, and the feet were white. A total of 11 VaJ /VaJ , 12 VaJ /+, and 12 +/+ mice aged between 14 and 53 days were used for electrophysiology, and these plus a further 8 mice (32 days to 8 months old) were used for histological studies. Mice were anaesthetised with urethane, a tracheal cannula was inserted, the middle ear was opened and a te£on-coated silver wire recording electrode was placed on the round window. A di¡erential reference electrode was placed on the muscle just dorsal to the bulla. Pure tone stimuli were presented through a closed, calibrated sound system inserted into the external ear canal. For compound action potential (CAP) and summating potential (SP) recording, 200 shaped tonebursts (15 ms duration, 1 ms rise/fall time, 115
ms interstimulus interval) were presented and the responses ¢ltered (5 kHz low pass) and averaged. Thresholds for the visual detection of a response in the averaged waveform were established using 5 dB intensity increments above and below threshold. Thresholds were obtained for 3, 6, 12, 18, 24 and 30 kHz stimuli. For measurement of cochlear microphonics (CM), continuous pure tones, stepping in frequency from 2 to 30 kHz, were presented as constant intensity sweeps up to 100 dB SPL in 10-dB steps. CM amplitudes were measured using a computercontrolled lock-in ampli¢er, which e¡ectively detects a frequency-speci¢c response in the presence of noise. Noise levels in the mouse can mask or mimic cochlear microphonics at low response amplitudes, so control recordings were taken with the bias current of the stimulus microphone turned o¡ to attenuate the sound output and assess the level of electrical noise in the system, which varied between mice. Following recording of cochlear responses, the endocochlear potential (EP) was measured. A small hole was made in the bone over the basal turn of the cochlear duct and a micropipette electrode ¢lled with 150 mM KCl was inserted through the lateral wall into scala media. The resting potential was recorded with respect to a silver/silver chloride reference electrode under the dorsal skin. Only stable recordings were included. All experiments on animals were conducted in full accordance with Home O¤ce regulations. After the physiological recordings, the cochleas were perfused through the round and oval windows with precooled ¢xative. The specimens were ¢xed in 2.5% glutaraldehyde/2% paraformaldehyde bu¡ered with 0.1 M sodium phosphate (pH 7.2) for 3 h at 4³C. From 13 cochleas the left and right strias were micro-dissected and a drawing was made showing the positions of the electrode hole and the strial segments. Individual segments were mounted in glycerol on glass slides, photographed and then transferred back to 0.1 M phosphate bu¡er for overnight washing before further TEM processing. After primary ¢xation, intact cochleas were left overnight in the same bu¡er and post-¢xed in 1% phosphate bu¡ered osmium tetroxide. After decalci¢cation in bu¡ered 4.13% EDTA at 4³C for 6^10 days, the specimens were dehydrated through graded alcohols and propylene oxide, block stained with phosphotungstic acid and uranyl acetate and embedded in Araldite. Semi-thin sections through the mid-modiolar plane of intact cochleas and strial segments of dissected specimens were stained with toluidine blue and examined by light microscopy. Ultra-thin sections cut from the same blocks were picked up on uncoated copper grids and viewed in a Phillips 410 transmission electron microscope operated at 80 kV. The distribution of pigment in the vestibular region of the cochlea was noted in just 2 mice of each geno-
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type. Fragments of the crus commune were then processed for TEM (as described above). 3. Results 3.1. Electrophysiology Both homozygous and heterozygous Varitint-waddler-J mutants show impaired cochlear responses. Furthermore, many of these mutants also show abnormally low endocochlear potentials, suggesting abnormal stria vascularis function. There is variability between individual mutants, as shown in Table 1A,B, but the homozygotes are more severely a¡ected than the heterozygotes. Endocochlear potential measurements are shown in Fig. 1. Normal mice show a gradual increase in EP up to about two weeks after birth (e.g. Steel and Barkway, 1989), and some further increase in EP after 14 days is seen in recordings from control mice used in this study. Some of the heterozygotes show EPs within the normal range, but at least three have abnormally low levels. However, the majority of the homozygotes have small or absent potentials. Cochlear microphonics re£ect primarily outer hair cell activity in the basal turn (i.e. in the region closest
Fig. 1. Endocochlear potential plotted as a function of age in days. In control mice, EP is still maturing to adult levels up to around 16^20 days. Some of the heterozygotes and most of the homozygotes show abnormally low EP levels. In one homozygote at 53 days old EP was recorded from both left and right ears (arrows), and was 0 mV in the left ear and 45 mV in the right ear.
to the recording electrode). In the control mice, increased input gave increased response amplitudes, with some sign of saturation at high frequencies (100
Table 1 A: Negative summating potentials and cochlear microphonics in VaJ /+ mice Age (days)
EP (mV)
14 14 16 16 18 18 18 20 20 32 42 42
77 82 59 80 33 74 87 0 0 90 87
Threshold for 3SP in dB SPL
Cochlear microphonics
3 kHz
6 kHz
12 kHz
18 kHz
24 kHz
30 kHz
87 92 92 85 97 85+ 102 NR 92+ NR NR NR
97 97 100 100+ 97+ 87+ 107 NR 97+ NR NR NR
105 100 102+ 95+ 97+ 90+ NR NR 92+ 127 125 127
125 105 105+ 95 97+ 87+ 127 NR 97+ 127 123 127
NR NR 107+ NR 97+ 90+ NR NR 117 124 127 127
NR NR 115 120 100 102+ NR NR NR NR NR NR
Up to 5 kHz Up to 22 kHz Up to 12 kHz Over whole range 2^30 kHz NR Up to 10 kHz NR NR Up to 20 kHz NR NR NR
NR NR 127 NR 124 NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
B: Negative summating potentials and cochlear microphonics in VaJ /VaJ mice 14 14 16 18 20 20 32 36 42 53 53
68 44 59 0 0 3 18 29 0
NR NR 112 NR NR NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
NR NR 123 130 125 125 NR NR NR NR NR
NR 125 123 125 125 125 NR NR 122 NR NR
NR: No response; +: positive SP present at high intensities; cochlear microphonics: range of frequencies from which a CM over 1 WV and over the background noise level could be detected.
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dB SPL). Among the heterozygous mutants, half gave some sign of a CM, mostly at low frequencies only (Table 1A), and these responses were always smaller than any recorded from a control mouse. The remaining six heterozygotes showed no CM above background noise levels. None of the homozygous mutants showed any sign of a CM response up to the maximum sound level used, 100 dB SPL. Compound action potentials re£ect the summed, synchronous activity of the cochlear nerve, and thresholds for detecting a CAP in the control group ranged from a mean of around 38^40 dB SPL at 12 and 18 kHz to around 60 dB SPL at 3 kHz, which is quite normal for a laboratory mouse. There was no obvious change in threshold with age over the age range studied. No mutants, either homozygotes or heterozygotes, showed any detectable CAP, even with stimuli up to 130 dB SPL. Summating potentials are dc o¡sets in the response waveform sustained for the duration of the toneburst, and can be positive (upwards in Fig. 2) or negative (downwards) in polarity. They correspond to the depolarisation of the hair cells during sound stimulation. Previous studies of SP in the mouse suggest that positive SPs re£ect activity of basal turn hair cells while negative SPs are derived from hair cell activity in the apical turn, in the normal mouse (Dallos et al., 1972 ; Dallos, 1986; Harvey and Steel, 1992). Some examples of normal SPs from a control mouse are shown in Fig. 2A. Negative SPs tend to be obtained at low frequencies and low intensities of stimulus, while positive SPs are recorded in response to high frequencies and high intensities. Both homozygotes and heterozygotes gave some SP responses at high stimulus intensities, suggesting that their hair cells can depolarise in response to sound. Examples of the waveforms from each genotype (the `best' mouse in each group) are shown in Fig. 2B,C. Thresholds were variable between animals, and
so are shown individually in Table 1. There are several general features to note about these responses. Firstly, homozygotes generally show higher thresholds for a negative SP and show more mice with no responses at
C Fig. 2. Some examples of waveforms recorded from (A) +/+ 42 days; (B) VaJ /+ 18 days, EP 74 mV; (C) VaJ /VaJ 16 days, EP 59 mV. Each waveform is a computer-averaged recording of 40 ms after the onset of the 15-ms toneburst, and the vertical scale varies because the waveforms have been plotted to a constant height to facilitate comparison of response shapes. Upwards de£ections represent positive voltage changes. Each column represents a single frequency, and rows represent di¡erent intensities of stimulus. The CAP is the sharp negative de£ection seen at the start of the toneburst in the control mouse in A. The SP is the o¡set in the response sustained for the 15-ms duration of the toneburst, and can be positive or negative. Positive SPs are seen at high frequencies and high intensities, while negative SPs are seen at low frequencies and low intensities. Both +SP and 3SP can be observed in the same waveform in some cases, because they show slightly di¡erent latencies; for example, the 12-kHz responses in A show a 3SP at low intensities with a +SP superimposed as intensity is increased to 100 dB SPL. This feature can also be seen in the heterozygote in B at all frequencies but a +SP was never seen in the homozygotes.
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Fig. 3. Strial surface preparations of the middle cochlea turns from (A) +/+ 42 days; (B) VaJ /VaJ 14 days. Pigment clusters (arrow heads) indicative of melanocytes are evenly distributed between the capillaries (arrows) of the control. The stria of the mutant shows a transition (*) from a vascularized to non-vascularized region. Subsequent EM examination of this tissue revealed melanocytes only in the vascularized region. Scale bars = 100 Wm.
all compared with heterozygotes. Secondly, homozygotes show no positive SP, while several of the heterozygotes have a positive SP component to their response waveform, as illustrated in Fig. 2B. Thirdly, mice aged 20 days and younger tended to give better responses than the older mice in both genotype groups. 3.2. Anatomy All control and heterozygote mice appear to have a normally pigmented stria vascularis. Pigment clumps observed from strial surface preparations (Fig. 3A) correspond to the melanocytes (light and dark intermediate cells) detected with electron microscopy (Fig. 4A,B). Light intermediate cells, the most common strial melanocyte, have a central position in the stria and their electron-lucent cytoplasm contains mitochondria and Golgi bodies. Dark intermediate cells have a peripheral pycnotic nucleus and large aggregates of an electrondense polymorphic matrix containing melanin granules (Cable and Steel, 1991). The majority of VaJ /VaJ mice have reduced, or no, pigmentation in the stria. Melanocytes in the mutants are distributed in bands along the stria so that in each cochlear turn there is usually a transition from unpigmented to pigmented tissue (Fig. 3B). There is no clear boundary in this transition zone; the large capillaries characteristic of the pigmented regions gradually reduce in diameter and abundance towards the unpigmented region. The distribution of pigment along the entire length of the stria was only measured in 2 homozygote mice (no. 34 and 3R): 35
and 49% of the length, respectively, contained melanocytes. The typical £attened ultrastructural appearance of a mutant stria with no melanocytes is shown in Fig. 4C. The marginal cells lie directly on the basal cells and no melanocytes are present. In many mutants with no obvious melanocytes, short marginal cell processes packed with mitochondria surround capillaries (Fig. 4D). Light intermediate cells (one type of melanocyte) Table 2 Pigment distribution in the stria vascularis of VaJ /VaJ mice No. 31 34 10 7 8 40 41b 27b 2 3L 3R a
Age (days) 14 14 18 20 20 32 32 42 53 53 53
EP (mV) 68 44 0 0 3 18 33 16 29 0 45
Cochlear turna AT
LAT
MT
BT
+ R 3 3 3 NE R 3 R 3 R
+ R 3 3 3 3 NE 3 R 3 R
+ R 3 3 3 3 NE 3 3 3 R
NE R 3 R NE NE R 3 3 3 R
The presence (+), reduction (R) or absence (3) of pigment in the apical (AT), lower apical (LAT), middle (MT) and basal turn (BT) of the cochlea was assessed from surface strial preparations, toluidine blue semi-thin sections and/or ultrathin sections. NE, tissue not examined. Strial pigment was present in all cochlear turns of +/+ and VaJ / + mice. In the majority of homozygous mutant ears, there was a precise correlation between presence/absence of an EP and presence/ absence or reduction in pigment. The results obtained from 2 mice (b ) might have arisen from technical limitations (see text).
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are present in the transitional pigmented regions (Fig. 4E). The fully pigmented regions of the mutant stria are similar in appearance to control tissue, the marginal cell
processes are well developed and there are no obvious structural abnormalities (Fig. 4F). There is a close correlation between the presence of
Fig. 4. Normal stria vascularis of (A) +/+ 18 days; (B) +/+ 36 days. Extensive marginal cell (MC) processes interdigitate with light (lc) and dark (dc) intermediate cells, and capillaries (ca). Basal cells (bc) separate the SV from the underlying spiral ligament (Sl). Unpigmented stria of (C) VaJ /VaJ 20 days; (D) VaJ /VaJ 36 days. Marginal cells bordering the scale media are only separated from the basal cells by capillaries. Marginal cell processes occasionally contain mitochondria (mi). (E) VaJ /VaJ 32 days, at the transition from unpigmented to pigmented region of the lower apical turn (F). Scale bars = 4 Wm.
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Fig. 5. Melanocytes in the crus commune of the vestibular region from (A) +/+ 4 months; (B) VaJ /VaJ 2 months. Melanosomes (arrow heads) clustered around the melanocyte nucleus underlie the basal lamina (bl) and dark cells (dk) of the crus commune. Scale bars = 1 Wm.
strial pigment and the presence of an EP in most of the mice studied. However, pigment distribution from many of the specimens shown in Table 2 was assessed by examination of sectioned cochleas so bands of pigmentation or reduced pigmentation may have gone unrecorded. This may explain the recording of a VaJ /VaJ mouse with an EP of 16 mV but no detected strial pigment. Flat strial surface preparations are the optimum way of recording the distribution of pigment along the entire length of the cochlea (see Fig. 7 in Cable et al., 1994), but we only made these preparations for 6 of the 19 mutants studied because we wanted to examine the whole cochlea in the remaining 13 mutants. Another VaJ /VaJ mutant (no. 41) with no EP had some pigment in the apical and basal turns, and a young VaJ / + (20 days old) with no EP appeared to have a normally pigmented stria. Even if part of this cochlea had reduced strial pigment, it is surprising that no EP was recorded. However, we did not section the entire cochlea of this specimen to check that the electrode was correctly positioned (e.g. Steel et al., 1987) and it is also possible that during EP measurement the tip of the electrode did not actually penetrate the scala media. The EP and distribution of strial pigment was only recorded from both ears in one animal. In this mature VaJ /VaJ mouse there was an asymmetrical pattern of
pigmentation : the unpigmented left ear had no EP whereas the right ear had reduced strial pigment and an EP of 45 mV. The distribution of melanocytes in the vestibular region of +/+, VaJ /+ and VaJ /VaJ is similar to that previously described in normally pigmented mice (Cable et al., 1994). There is no obvious ultrastructural di¡erence in vestibular melanocytes from controls and mutants (Fig. 5). Premelanosomes and melanosomes in the crus commune are slightly more spherical than those in the stria. The organ of Corti appeared normal in all control mice (Figs. 6A and 7A). Some degeneration of hair cells was present in all VaJ /+ mice but there was great variation in the number of abnormal cells and they tended to be scattered along the cochlea rather than restricted to speci¢c regions. Abnormalities of inner hair cells (IHC) were present in 14 day old VaJ /+ mice (the youngest animals examined in this study). Inner hair stereocilia were often fused together and reduced in number (Fig. 6C), or occasionally, completely absent. The cell cytoplasm was atypically electron-lucent and the apical surface of some IHC was swollen; the cuticular plate and surrounding belt of cytoplasm bulged out into the scala media (Fig. 6D). Fragmentation of these convex cuticular plates was common. In older mice,
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synaptic contacts at the base of the cells were rarely seen. Generally, outer hair cells (OHC) showed fewer signs of degeneration compared to the IHC with the
exception of one heterozygote (no. 16 that had no EP) in which many of the OHC had completely degenerated. Other OHC in VaJ /+ mice 18 days and older
Fig. 6. Inner hair cells in the middle cochlear turn from (A) +/+ 32 days; (B) VaJ /VaJ 36 days. Stereocilia (arrow) are missing from the mutant and the entire apical surface of the cell is swollen. (C) VaJ /+ 14 days, fused stereocilia (arrow head). Degenerating IHC, reduced in length with distorted apical surfaces, in (D) VaJ /+ 42 days; (E) VaJ /VaJ 18 days. Scale bars = 4 Wm.
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Fig. 7. Outer hair cells in the middle cochlear turn of (A) +/+ 18 days; (B) VaJ /VaJ 18 days; (C) VaJ /VaJ 20 days. Fused stereocilia (arrows), rounded vacuolated cells are characteristic of VaJ OHC degeneration. Scale bars = 4 Wm.
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had fused stereocilia. All supporting cells and Reissner's and tectorial membranes appeared una¡ected in the heterozygotes. Severe atrophy of the organ of Corti characterised the cochleas of VaJ /VaJ mice. IHC, when present, always had a swollen apical surface with an underlying thickened terminal web. Stereocilia were absent (Fig. 6B). Most IHC exhibited signs of degeneration (Fig. 6E) and contact with a¡erent nerve endings was rare. A few OHC retained their elongate shape with a basally positioned nucleus (Fig. 7B) but the majority consisted of condensed rounded cells with electron-lucent cytoplasm (Fig. 7C). Nuclei were intact often surrounded by lipofuscin granules but no e¡erent synapses were detected at the base of the cells. Stereocilia at the cell surface were fused together or absent. These IHC and OHC abnormalities present 14 days after birth became more widespread along the cochlea with age and were not restricted to any particular region of the cochlea. In mice 20 days and older, hair cells in some regions were completely atrophic. Elsewhere most OHC were shrunken, with just a few elongate cells remaining in the apical cochlea turn. IHC were even more sparsely distributed and their supporting cells were also degenerate. In all of the mice 53 days and younger (including those VaJ /VaJ mice with a reduced or absent EP), Reissner's membrane remained in its normal position. Similarly, no abnormalities of the tectorial membrane or spiral prominence were identi¢ed. In the oldest mouse examined (8 months old), atrophy of the hair and pillar cells was complete, just fragments of individual supporting cells remained. 4. Discussion The physiological and structural studies reported here suggest that the Varitint-waddler-J mutant has an unusual combination of primary defects in both the neuroepithelium and the stria vascularis. The stria defect is variable, and in extreme cases there is no recordable EP associated with an absence of melanocytes in the stria. We have observed a similar strial pathology in other mouse mutants with coat colour defects (Cable et al., 1992, 1994) and the ¢ndings strongly suggest that the lack of melanocytes in the stria causes strial dysfunction and a reduced EP. We do not know the reason for the lack of melanocytes, and it may be that the developing stria has a primary abnormality which directly a¡ects its ability to produce an EP as well as making it an inhospitable environment for survival of melanocytes. However, as we already know that melanocytes are essential for normal strial function, and that skin melanocytes are a¡ected by the VaJ mutation, a simpler explanation of a reduced EP is that the mutation causes the stria defect by its e¡ects on melanocytes.
It seems unlikely that the organ of Corti defects and the impaired cochlear responses in VaJ mutants are all a consequence of a reduced EP for several reasons. Firstly, in some mutants the EP (as recorded in the basal turn) was within the normal range but there was still a severe impairment in responses to sound. Secondly, in W and Sl mutants, which we have studied extensively, there is often no EP but hair cells survive and look normal for several weeks before they start to degenerate (Steel and Harvey, 1992 ; Steel et al., 1987); the hair cell defects seen in VaJ are more severe than this, suggesting that they may be a¡ected directly by the mutation. It is also unlikely that the strial defects are a consequence of a primary organ of Corti defect because in several neuroepithelial mutants we have studied, such as dn and je, there are severe hair cell abnormalities and a complete lack of cochlear responses, but the EP remains normal even in very old mice (Bock and Steel, 1983 ; Steel and Bock, 1983b). In some VaJ mice as young as 20 days, the stria is atrophic. There is no evidence to suggest that collapse of Reissner's membrane might be responsible for the reduced or absent EP in VaJ mice, although this characteristic has been reported as a secondary e¡ect of cochleo-saccular degeneration (e.g. Deol, 1970 ; Mair, 1973). For these reasons, we suggest that the VaJ mutation most likely affects both the stria and the organ of Corti directly. The lack of any detectable CAP in the mutants correlates with the lack of any behavioural response to sound. It suggests that little or no responses to sound are passed to the brain. However, the ¢nding of an SP in some of the homozygotes is interesting, because it suggests that, at least in some cases, the mutant hair cells can depolarise, and that the non-linear response of the hair cell to stimulation is preserved. The SPs observed in homozygotes are all negative, and in a control mouse negative SPs are thought most likely to originate from apical turn hair cell activity. Hence, this might indicate that hair cells in the apex might be responsible for the negative SP seen, correlating with the ¢nding that the apical turn organ of Corti is better preserved than the basal turn. However, in the pathological cochleas studied, and at the very high sound levels used to elicit the SP responses, it is probably not appropriate to assume that the responses recorded are generated at the same part of the cochlear duct as might be the case in normal mice. In the mutant mice, the frequency/place map and the balance between responses of di¡erent hair cell groups are likely to be abnormal. Ultrastructural studies of the VaJ cochlea revealed a progressive degeneration of the organ of Corti with age. However, hearing loss in these mice is already advanced 14 days after birth. At this age, the observed hair cell abnormalities (such as fused stereocilia, swollen cuticular plates) may not be su¤cient in themselves to account for the poor physiological responses. It is more
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likely that some biochemical alteration of the cells is responsible for hearing loss in young mice. In a previous study of the other allele of Varitint-waddler, Va, hair cells were studied in surface preparations following staining for succinic dehydrogenase activity (Steel, 1978). Control incubations suggested that the technique is relatively non-speci¢c and that substrates other than succinate may give a strong labelling reaction, and tissue permeability to the reagents may also a¡ect labelling (e.g. Steel and Bock, 1983b). Nonetheless, in normal cochleas the hair cells are strongly labelled compared with the weak labelling of the supporting cells. In a total of 55 Va/+ cochleas ranging from 10 to 100 days old, compared with 42 age-matched littermate controls, progressive hair cell degeneration was seen in the mutants. Furthermore, at the youngest stages, 10 and 15 days old, most of the hair cells were present but only a small proportion of them showed the normal strong labelling, scattered around the organ of Corti, while the rest were unlabelled. This suggests that there may be an early physiological abnormality of these hair cells preventing either their uptake or their reduction of the Nitro Blue Tetrazolium to form insoluble formazan, the basis of the labelling reaction. If a similar disruption in reagent uptake or dehydrogenase activity is present in VaJ mice this may explain the rapid degeneration of hair cells. The observed scattered loss of hair cells along the cochlea in VaJ mice 20 days and older is commonly observed in other mouse mutants such as dn and je (Steel, 1978; Steel and Bock, 1983b). This genetic abnormality is distinct from the localized patches of damage found in animals exposed to excess noise (e.g. Johnsson and Hawkins, 1976). Earlier work on the Va allele suggested that it is likely to be more severe than the VaJ allele in its e¡ect on the ear, because the coat colour defect is more extreme in Va and there are clear vestibular defects in Va but no obvious behavioural defects in VaJ (Cloudman and Bunker, 1945 ; Deol, 1954; Lane, 1972). It does appear from the current study that the pathology of the inner ear of VaJ is less extreme compared to that reported in Va mice. Steel and Harvey (1992) made a distinction between those neuroepithelial mutants with auditory responses (such as shaker-1) and those without (such as deafness); VaJ mice fall into the former category and Va into the latter. However, it is not possible from the published accounts to make detailed comparisons of the physiology of Va and VaJ ears. Mikaelian et al. (1965) found no responses at all in Va, but were not clear whether they were heterozygotes or homozygotes, what age they were, or the maximum SPL used. Berlin et al. (1969) found abnormal vocalisations in Va mice and suggested that this was associated with complete deafness in these mutants. However, again it is not clear whether Va/Va or Va/+ mice were used in this study. Detailed light microscope examination of Va
135
cochleas was undertaken by Deol (1954) and Kocher (1960). They reported progressive degeneration of the stria from 3 weeks onwards in heterozygotes, and a particularly thin stria in homozygotes from an early stage. Degeneration of hair cells in Va mice was apparent from 11 days onwards and progressed rapidly with age. Hair cell loss was variable and often asymmetric but was more advanced than that observed by us in VaJ mice. For example, hair cell degeneration was reported to be complete in one 35 day old Va/Va mouse (Deol, 1954). Abnormalities of the tectorial membrane present in immature Va were not observed during the current ultrastructural study of VaJ mice. However, pigmentation defects are present in the cochleas of Va and VaJ . Deol (1954) and Kocher (1960) both reported strial defects in Va mice as early as 12 days after birth. The variable thickness of the stria reported in Va/Va mice (Deol, 1954) may be indicative of the spotting observed here in VaJ /VaJ mice. Deafness is often associated with pigmentation anomalies in humans and other mammals including mice, rats, mink, cats and dogs (e.g. Cable et al., 1994 ; Hageman and Delleman, 1977 ; Johnsson and Hawkins, 1976; Mair, 1973 ; Sugiura and Hilding, 1970 ; Tsujimura et al., 1991). In addition to the disrupted pigmentation patterns in the skin or coat, these animals exhibit spotting of internal organs. Spotting genes cause a lack of pigment cells rather than acting by disrupting melanin biosynthesis which occurs in the melanocytes of albinos. In spotting mutants, reduced strial pigmentation indicating a lack of melanocytes correlates precisely with reduced EP. Although little progress has been made over recent years in assessing the precise function of melanocytes within the stria (Schulte and Steel, 1994), there is an increasing awareness of the similarities of these cells in the skin and cochlea. Conlee et al. (1994) demonstrated that melanocytes in the stria undergo proliferation at a similar rate to those in the skin and suggested that the factors known to a¡ect replication and melanogenesis of skin pigment cells may also a¡ect those in the ear. Unilateral e¡ects are common in spotting, and so only one ear may be a¡ected (e.g. Cable et al., 1994; Bosher and Hallpike, 1965). In this study, both ears were examined in only one mutant but this example was su¤cient to demonstrate the presence of unilateral spotting in the cochleas of VaJ mice. A preliminary examination of the vestibular region revealed a normal distribution of melanocytes. However, insu¤cient specimens were examined to determine whether or not the spotting observed in the cochlea also occurred in other regions of the inner ear. In contrast to other spotting mutants previously examined, strial pigment in VaJ /VaJ mice is not always con¢ned to a single region of the cochlea but may be present in several pigmented patches. This distribution of strial pigmentation may
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be related to the unusual coat pattern of these mice. A combination of spotting and dilution in the coat of Va mice led Silvers (1979) to suggest a complex Va-locus. VaJ apparently arose from Va, which might suggest a partial reversion. Clearly, identi¢cation of this gene will shed important light on the genetics of pigmentation, in addition to revealing the molecular basis of this dual cochleo-saccular/neuroepithelial hearing loss. Acknowledgments We thank Mike Edel for technical assistance and N. Glenn for animal care. References Berlin, C.I., Majeau, D.A., Steiner, S., 1969. Hearing and vocal output in normal, deaf and intact mice. J. Aud. Res. 9, 318^331. Bock, G.R., Steel, K.P., 1983. Inner ear pathology in the deafness mutant mouse. Acta Otolaryngol. 96, 39^47. Bosher, S.K., Hallpike, C.S., 1965. Observations on the histological features, development and pathogenesis of the inner ear degeneration of the deaf white cat. Proc. R. Soc. London B 162, 147^170. Cable, J., Steel, K.P., 1991. Identi¢cation of two types of melanocyte within the stria vascularis of the mouse inner ear. Pigment Cell Res. 4, 87^101. Cable, J., Barkway, C., Steel, K.P., 1992. Characteristics of stria vascularis melanocytes of Viable Dominant Spotting (Wv /Wv ) mouse mutants. Hear. Res. 64, 6^20. Cable, J., Huszar, D., Jaenisch, R., Steel, K.P., 1994. E¡ects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse. Pigment Cell Res. 7, 17^32. Cloudman, A.M., Bunker, L.E., 1945. The varitint-waddler mouse. A dominant mutation in Mus musculus. J. Hered. 36, 259^263. Conlee, J.W., Gerity, L.C., Bennett, M.L., 1994. Ongoing proliferation of melanocytes in the stria vascularis of adult guinea pigs. Hear. Res. 79, 115^122. Cools, A.R., 1972a. Neurochemical correlates of the waltzing-shaker syndrome in the varitint-waddler mouse. Psychopharmacologia 24, 384^396. Cools, A.R., 1972b. Asymmetrical spotting and direction of circling in the varitint-waddler mouse. J. Hered. 63, 161^171. Dallos, P., 1986. Neurobiology of inner and outer hair cells: intracellular recordings. Hear. Res. 22, 185^198. Dallos, P., Schoeny, Z.G., Cheatham, M.A., 1972. Cochlear summating potentials: Descriptive aspects. Acta Otolaryngol. (Suppl.) 302, 1^46. Deol, M.S., 1954. The anomalies of the labyrinth of the mutants Varitint-Waddler, Shaker-2 and Jerker in the mouse. J. Genet. 52, 562^588.
Deol, M.S., 1970. The origin of the acoustic ganglion and e¡ects of the gene dominant spotting (Wv ) in the mouse. J. Embryol. Exp. Morphol. 23, 773^784. Hageman, M.J., Delleman, J.W., 1977. Heterogeneity in Waardenburg Syndrome. Am. J. Hum. Genet. 29, 468^485. Harvey, D., Steel, K.P., 1992. The development and interpretation of the summating potential response. Hear. Res. 61, 137^146. Johnsson, L.G., Hawkins, J.E., 1976. Degeneration patterns in human ears exposed to noise. Ann. Otol. Rhinol. Laryngol. 85, 725^739. Kocher, W., 1960. Untersuchungen zur genetik und pathologie der entwicklung von 8 labyrinthmutanten (Deaf-Waltzer-Shaker-mutanten) der maus (Mus musculus). Z. Verebungslehre 91, 114^140. Lane, P.W., 1972. Two new mutations in linkage group XVI of the house mouse, Flaky tail and Varitant-J. J. Hered. 63, 355-140. Mair, I.W.S., 1973. Hereditary deafness in the white cat. Acta Otolaryngol. (Suppl.) 314, 5^48. Mikaelian, D.O., Alford, B.R., Ruben, R.J., 1965. Cochlear potentials and VIII nerve action potentials in normal and genetically deaf mice. Ann. Otol. Rhinol. Laryngol. 74, 146^157. Schulte, B.A., Steel, K.P., 1994. Expression of alpha-subunit and beta-subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the WV or Sld loci. Hear. Res. 78, 65^76. Silvers, W.K., 1979. The Coat Colors of Mice: a Model for Gene Action and Interaction. Springer, New York, NY. Steel, K.P., 1978. Studies on Mice with Genetical and Experimentallyinduced Abnormalities of the Inner Ear. Ph.D. Thesis. University of London. Steel, K.P., 1991. Similarities between mice and humans with hereditary deafness. Ann. NY Acad. Sci. 630, 68^79. Steel, K.P., 1995. Inherited hearing defects in mice. Annu. Rev. Genet. 29, 675^701. Steel, K.P., Barkway, C., 1989. Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear. Development 107, 453^463. Steel, K.P., Brown, S.D.M., 1994. Genes and deafness. Trends Genet. 10, 428^435. Steel, K.P., Bock, G.R., 1983a. Hereditary inner-ear abnormalities in animals. Arch. Otolaryngol. 109, 22^29. Steel, K.P., Bock, G.R., 1983b. Cochlear dysfunction in the Jerker mouse. Behav. Neurosci. 97, 381^391. Steel, K.P., Harvey, D., 1992. Development of auditory function in mutant mice. In: Romand, R. (Ed.), Development of Auditory Systems 2. Elsevier, Amsterdam, pp. 221^242. Steel, K.P., Barkway, C., Bock, G.R., 1987. Strial dysfunction in mice with cochleo-saccular abnormalities. Hear. Res. 27, 11^26. Sugiura, A., Hilding, D.A., 1970. Cochleo-saccular degeneration in Hedlund white mink. Acta Otolaryngol. 69, 126^137. Tsujimura, T., Hirota, S., Nomura, S., Niwa, Y., Yamazaki, M., Tono, T., Morii, E., Hyung-Min, K., Kondo, K., Nishimune, Y., Kitamura, Y., 1991. Characterisation of Ws mutant allele of rats: A 12-base deletion in tyrosinase domain of c-kit gene. Blood 78, 1942^1946.
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Combined cochleo-saccular and neuroepithelial abnormalities in the Varitint-waddler-J (VaJ ) mouse Joanne Cable a
a;b;
*, Karen P. Steel
a
MRC Institute of Hearing Research, University Park, Nottingham, NG7 2RD, UK b School of Biological Sciences, University of Bristol, Bristol, BS8 1UG, UK Received 21 January 1998; revised 18 May 1998; accepted 23 May 1998
Abstract Hearing loss in Varitint-waddler-J (VaJ ) mice is of mixed origin with both cochleo-saccular and neuroepithelial components. Both VaJ /VaJ and VaJ /+ mutants show impaired cochlear function, but the homozygotes are more severely affected than heterozygotes. Neither group have any detectable compound action potential. Cochlear microphonics are only seen in half of the heterozygotes, at a reduced amplitude and raised threshold, and are not detected in any homozygotes. Summating potentials (SP) responses are seen in most of the heterozygotes, at high stimulus levels. The only responses in homozygotes were negative SPs seen in half of the mutants at very high sound levels, while the remaining homozygotes showed no responses to sound stimulation. Endocochlear potentials (EP) were often small or absent in both groups of mutants, with the homozygotes being more severely affected. Reduced pigmentation in the stria vascularis appears to be associated with a reduced EP, while a primary defect of the neuroepithelium, detectable by electron microscopy in hair cells of 14 day old mice, dramatically influences evoked potentials. z 1998 Elsevier Science B.V. All rights reserved. Key words: Endocochlear potential; Stria vascularis; Melanocyte; Compound action potential; Summating potential; Mouse mutant; Pigment defect
1. Introduction Numerous mouse mutants with hearing defects are available as models for understanding human deafness (Steel, 1991, 1995). Functional and anatomical studies of the mouse and human inner ear have identi¢ed the same broad categories of pathology in the two species : morphogenetic, cochleo-saccular and neuroepithelial. Morphogenetic abnormalities involve gross structural deformities of the labyrinth. A strial abnormality is the primary cochleo-saccular defect in which there is a reduced or absent EP and sometimes collapse of Reissner's membrane, which eventually results in degeneration of the organ of Corti and spiral ganglion cells. Neuroepithelial defects originate in the organ of Corti and do not a¡ect the stria directly (Steel and Bock, 1983a). * Corresponding author. School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK. Tel.: +44 (117) 9287473; Fax: +44 (117) 9257374; E-mail: [email protected]
Physiological and structural analyses of all these mouse mutants provides important information on the biological basis of normal hearing and genetic deafness. Unravelling the complexity of the inner ear has progressed more recently to identifying genes and their mutations which a¡ect normal hearing (Steel and Brown, 1994). To date, 41 loci have been identi¢ed that are involved in non-syndromic deafness (Hereditary Hearing Impairment homepage http://dnalab.www.uia.ac.be/dnalab/ hhh/). The full value of data emerging on the genetics of deafness derived from studies of mouse mutants and their human homologues will be maximized if it can be examined in the light of known functional and structural abnormalities of a particular mouse mutant. The Varitint-waddler-J mouse (VaJ ) arose as a semidominant mutation on chromosome 12 from the Va allele (Lane, 1972; Silvers, 1979). The Va mouse was ¢rst observed by Cloudman and Bunker (1945) in an outcross between C57 black and C57 brown stocks. Va/
0378-5955 / 98 / $19.00 ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 5 9 5 5 ( 9 8 ) 0 0 1 0 7 - 5
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+ mice display a combination of spotting with dilution of coat colour and exhibit near normal viability. The homozygotes are white except for small patches of coloured hairs on the ears and tail base. Va/Va males are sterile and many mice of this genotype die before birth. All Va mice are deaf and show circling behaviour, head tossing and hyperactivity (Cools, 1972a,b). Deol (1954) examining the inner ear morphology of Va/+ mice noted the ¢rst defects 4 days after birth. Anomalies of the tectorial membrane, followed by abnormalities in the spiral ganglion, organ of Corti and then strial atrophy are seen in the Va heterozygotes and homozygotes, characteristic of a neuroepithelial defect. The e¡ects of the VaJ mutation appear to be less severe than those associated with Va. VaJ /+ have only a slightly diluted coat colour (resembling Wv /+ mice). Both VaJ /+ and VaJ /VaJ are deaf but they behave normally and are fertile although a number of homozygotes die in utero (Lane, 1972). This study was undertaken to examine the physiological and structural abnormalities of the inner ear of VaJ mice. 2. Materials and methods Mice carrying the VaJ allele on a C57BL/6/C3H F1 genetic background were obtained from the Jackson Laboratory, and a breeding colony established without the Hd mutation which originally was segregating in the supplied stock. Heterozygous mice were intercrossed to generate VaJ /VaJ , VaJ /+ and +/+ mice, easily distinguished by their coat colours. All control mice had a black coat, pigmented pinna, tail, and feet. Heterozygotes had a grey diluted coat colour and a white belly spot often with an associated black patch. Their feet were white but the tails were at least partially pigmented. Homozygotes had a white dorsal coat with grey £ecks and some black patches. The typical white ventral surface had occasional grey patches. Pigmented patches when present on the head were often asymmetrical. The pinna and tail were pigmented or partially pigmented, and the feet were white. A total of 11 VaJ /VaJ , 12 VaJ /+, and 12 +/+ mice aged between 14 and 53 days were used for electrophysiology, and these plus a further 8 mice (32 days to 8 months old) were used for histological studies. Mice were anaesthetised with urethane, a tracheal cannula was inserted, the middle ear was opened and a te£on-coated silver wire recording electrode was placed on the round window. A di¡erential reference electrode was placed on the muscle just dorsal to the bulla. Pure tone stimuli were presented through a closed, calibrated sound system inserted into the external ear canal. For compound action potential (CAP) and summating potential (SP) recording, 200 shaped tonebursts (15 ms duration, 1 ms rise/fall time, 115
ms interstimulus interval) were presented and the responses ¢ltered (5 kHz low pass) and averaged. Thresholds for the visual detection of a response in the averaged waveform were established using 5 dB intensity increments above and below threshold. Thresholds were obtained for 3, 6, 12, 18, 24 and 30 kHz stimuli. For measurement of cochlear microphonics (CM), continuous pure tones, stepping in frequency from 2 to 30 kHz, were presented as constant intensity sweeps up to 100 dB SPL in 10-dB steps. CM amplitudes were measured using a computercontrolled lock-in ampli¢er, which e¡ectively detects a frequency-speci¢c response in the presence of noise. Noise levels in the mouse can mask or mimic cochlear microphonics at low response amplitudes, so control recordings were taken with the bias current of the stimulus microphone turned o¡ to attenuate the sound output and assess the level of electrical noise in the system, which varied between mice. Following recording of cochlear responses, the endocochlear potential (EP) was measured. A small hole was made in the bone over the basal turn of the cochlear duct and a micropipette electrode ¢lled with 150 mM KCl was inserted through the lateral wall into scala media. The resting potential was recorded with respect to a silver/silver chloride reference electrode under the dorsal skin. Only stable recordings were included. All experiments on animals were conducted in full accordance with Home O¤ce regulations. After the physiological recordings, the cochleas were perfused through the round and oval windows with precooled ¢xative. The specimens were ¢xed in 2.5% glutaraldehyde/2% paraformaldehyde bu¡ered with 0.1 M sodium phosphate (pH 7.2) for 3 h at 4³C. From 13 cochleas the left and right strias were micro-dissected and a drawing was made showing the positions of the electrode hole and the strial segments. Individual segments were mounted in glycerol on glass slides, photographed and then transferred back to 0.1 M phosphate bu¡er for overnight washing before further TEM processing. After primary ¢xation, intact cochleas were left overnight in the same bu¡er and post-¢xed in 1% phosphate bu¡ered osmium tetroxide. After decalci¢cation in bu¡ered 4.13% EDTA at 4³C for 6^10 days, the specimens were dehydrated through graded alcohols and propylene oxide, block stained with phosphotungstic acid and uranyl acetate and embedded in Araldite. Semi-thin sections through the mid-modiolar plane of intact cochleas and strial segments of dissected specimens were stained with toluidine blue and examined by light microscopy. Ultra-thin sections cut from the same blocks were picked up on uncoated copper grids and viewed in a Phillips 410 transmission electron microscope operated at 80 kV. The distribution of pigment in the vestibular region of the cochlea was noted in just 2 mice of each geno-
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type. Fragments of the crus commune were then processed for TEM (as described above). 3. Results 3.1. Electrophysiology Both homozygous and heterozygous Varitint-waddler-J mutants show impaired cochlear responses. Furthermore, many of these mutants also show abnormally low endocochlear potentials, suggesting abnormal stria vascularis function. There is variability between individual mutants, as shown in Table 1A,B, but the homozygotes are more severely a¡ected than the heterozygotes. Endocochlear potential measurements are shown in Fig. 1. Normal mice show a gradual increase in EP up to about two weeks after birth (e.g. Steel and Barkway, 1989), and some further increase in EP after 14 days is seen in recordings from control mice used in this study. Some of the heterozygotes show EPs within the normal range, but at least three have abnormally low levels. However, the majority of the homozygotes have small or absent potentials. Cochlear microphonics re£ect primarily outer hair cell activity in the basal turn (i.e. in the region closest
Fig. 1. Endocochlear potential plotted as a function of age in days. In control mice, EP is still maturing to adult levels up to around 16^20 days. Some of the heterozygotes and most of the homozygotes show abnormally low EP levels. In one homozygote at 53 days old EP was recorded from both left and right ears (arrows), and was 0 mV in the left ear and 45 mV in the right ear.
to the recording electrode). In the control mice, increased input gave increased response amplitudes, with some sign of saturation at high frequencies (100
Table 1 A: Negative summating potentials and cochlear microphonics in VaJ /+ mice Age (days)
EP (mV)
14 14 16 16 18 18 18 20 20 32 42 42
77 82 59 80 33 74 87 0 0 90 87
Threshold for 3SP in dB SPL
Cochlear microphonics
3 kHz
6 kHz
12 kHz
18 kHz
24 kHz
30 kHz
87 92 92 85 97 85+ 102 NR 92+ NR NR NR
97 97 100 100+ 97+ 87+ 107 NR 97+ NR NR NR
105 100 102+ 95+ 97+ 90+ NR NR 92+ 127 125 127
125 105 105+ 95 97+ 87+ 127 NR 97+ 127 123 127
NR NR 107+ NR 97+ 90+ NR NR 117 124 127 127
NR NR 115 120 100 102+ NR NR NR NR NR NR
Up to 5 kHz Up to 22 kHz Up to 12 kHz Over whole range 2^30 kHz NR Up to 10 kHz NR NR Up to 20 kHz NR NR NR
NR NR 127 NR 124 NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
B: Negative summating potentials and cochlear microphonics in VaJ /VaJ mice 14 14 16 18 20 20 32 36 42 53 53
68 44 59 0 0 3 18 29 0
NR NR 112 NR NR NR NR NR NR NR NR
NR NR NR NR NR NR NR NR NR NR NR
NR NR 123 130 125 125 NR NR NR NR NR
NR 125 123 125 125 125 NR NR 122 NR NR
NR: No response; +: positive SP present at high intensities; cochlear microphonics: range of frequencies from which a CM over 1 WV and over the background noise level could be detected.
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dB SPL). Among the heterozygous mutants, half gave some sign of a CM, mostly at low frequencies only (Table 1A), and these responses were always smaller than any recorded from a control mouse. The remaining six heterozygotes showed no CM above background noise levels. None of the homozygous mutants showed any sign of a CM response up to the maximum sound level used, 100 dB SPL. Compound action potentials re£ect the summed, synchronous activity of the cochlear nerve, and thresholds for detecting a CAP in the control group ranged from a mean of around 38^40 dB SPL at 12 and 18 kHz to around 60 dB SPL at 3 kHz, which is quite normal for a laboratory mouse. There was no obvious change in threshold with age over the age range studied. No mutants, either homozygotes or heterozygotes, showed any detectable CAP, even with stimuli up to 130 dB SPL. Summating potentials are dc o¡sets in the response waveform sustained for the duration of the toneburst, and can be positive (upwards in Fig. 2) or negative (downwards) in polarity. They correspond to the depolarisation of the hair cells during sound stimulation. Previous studies of SP in the mouse suggest that positive SPs re£ect activity of basal turn hair cells while negative SPs are derived from hair cell activity in the apical turn, in the normal mouse (Dallos et al., 1972 ; Dallos, 1986; Harvey and Steel, 1992). Some examples of normal SPs from a control mouse are shown in Fig. 2A. Negative SPs tend to be obtained at low frequencies and low intensities of stimulus, while positive SPs are recorded in response to high frequencies and high intensities. Both homozygotes and heterozygotes gave some SP responses at high stimulus intensities, suggesting that their hair cells can depolarise in response to sound. Examples of the waveforms from each genotype (the `best' mouse in each group) are shown in Fig. 2B,C. Thresholds were variable between animals, and
so are shown individually in Table 1. There are several general features to note about these responses. Firstly, homozygotes generally show higher thresholds for a negative SP and show more mice with no responses at
C Fig. 2. Some examples of waveforms recorded from (A) +/+ 42 days; (B) VaJ /+ 18 days, EP 74 mV; (C) VaJ /VaJ 16 days, EP 59 mV. Each waveform is a computer-averaged recording of 40 ms after the onset of the 15-ms toneburst, and the vertical scale varies because the waveforms have been plotted to a constant height to facilitate comparison of response shapes. Upwards de£ections represent positive voltage changes. Each column represents a single frequency, and rows represent di¡erent intensities of stimulus. The CAP is the sharp negative de£ection seen at the start of the toneburst in the control mouse in A. The SP is the o¡set in the response sustained for the 15-ms duration of the toneburst, and can be positive or negative. Positive SPs are seen at high frequencies and high intensities, while negative SPs are seen at low frequencies and low intensities. Both +SP and 3SP can be observed in the same waveform in some cases, because they show slightly di¡erent latencies; for example, the 12-kHz responses in A show a 3SP at low intensities with a +SP superimposed as intensity is increased to 100 dB SPL. This feature can also be seen in the heterozygote in B at all frequencies but a +SP was never seen in the homozygotes.
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Fig. 3. Strial surface preparations of the middle cochlea turns from (A) +/+ 42 days; (B) VaJ /VaJ 14 days. Pigment clusters (arrow heads) indicative of melanocytes are evenly distributed between the capillaries (arrows) of the control. The stria of the mutant shows a transition (*) from a vascularized to non-vascularized region. Subsequent EM examination of this tissue revealed melanocytes only in the vascularized region. Scale bars = 100 Wm.
all compared with heterozygotes. Secondly, homozygotes show no positive SP, while several of the heterozygotes have a positive SP component to their response waveform, as illustrated in Fig. 2B. Thirdly, mice aged 20 days and younger tended to give better responses than the older mice in both genotype groups. 3.2. Anatomy All control and heterozygote mice appear to have a normally pigmented stria vascularis. Pigment clumps observed from strial surface preparations (Fig. 3A) correspond to the melanocytes (light and dark intermediate cells) detected with electron microscopy (Fig. 4A,B). Light intermediate cells, the most common strial melanocyte, have a central position in the stria and their electron-lucent cytoplasm contains mitochondria and Golgi bodies. Dark intermediate cells have a peripheral pycnotic nucleus and large aggregates of an electrondense polymorphic matrix containing melanin granules (Cable and Steel, 1991). The majority of VaJ /VaJ mice have reduced, or no, pigmentation in the stria. Melanocytes in the mutants are distributed in bands along the stria so that in each cochlear turn there is usually a transition from unpigmented to pigmented tissue (Fig. 3B). There is no clear boundary in this transition zone; the large capillaries characteristic of the pigmented regions gradually reduce in diameter and abundance towards the unpigmented region. The distribution of pigment along the entire length of the stria was only measured in 2 homozygote mice (no. 34 and 3R): 35
and 49% of the length, respectively, contained melanocytes. The typical £attened ultrastructural appearance of a mutant stria with no melanocytes is shown in Fig. 4C. The marginal cells lie directly on the basal cells and no melanocytes are present. In many mutants with no obvious melanocytes, short marginal cell processes packed with mitochondria surround capillaries (Fig. 4D). Light intermediate cells (one type of melanocyte) Table 2 Pigment distribution in the stria vascularis of VaJ /VaJ mice No. 31 34 10 7 8 40 41b 27b 2 3L 3R a
Age (days) 14 14 18 20 20 32 32 42 53 53 53
EP (mV) 68 44 0 0 3 18 33 16 29 0 45
Cochlear turna AT
LAT
MT
BT
+ R 3 3 3 NE R 3 R 3 R
+ R 3 3 3 3 NE 3 R 3 R
+ R 3 3 3 3 NE 3 3 3 R
NE R 3 R NE NE R 3 3 3 R
The presence (+), reduction (R) or absence (3) of pigment in the apical (AT), lower apical (LAT), middle (MT) and basal turn (BT) of the cochlea was assessed from surface strial preparations, toluidine blue semi-thin sections and/or ultrathin sections. NE, tissue not examined. Strial pigment was present in all cochlear turns of +/+ and VaJ / + mice. In the majority of homozygous mutant ears, there was a precise correlation between presence/absence of an EP and presence/ absence or reduction in pigment. The results obtained from 2 mice (b ) might have arisen from technical limitations (see text).
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are present in the transitional pigmented regions (Fig. 4E). The fully pigmented regions of the mutant stria are similar in appearance to control tissue, the marginal cell
processes are well developed and there are no obvious structural abnormalities (Fig. 4F). There is a close correlation between the presence of
Fig. 4. Normal stria vascularis of (A) +/+ 18 days; (B) +/+ 36 days. Extensive marginal cell (MC) processes interdigitate with light (lc) and dark (dc) intermediate cells, and capillaries (ca). Basal cells (bc) separate the SV from the underlying spiral ligament (Sl). Unpigmented stria of (C) VaJ /VaJ 20 days; (D) VaJ /VaJ 36 days. Marginal cells bordering the scale media are only separated from the basal cells by capillaries. Marginal cell processes occasionally contain mitochondria (mi). (E) VaJ /VaJ 32 days, at the transition from unpigmented to pigmented region of the lower apical turn (F). Scale bars = 4 Wm.
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Fig. 5. Melanocytes in the crus commune of the vestibular region from (A) +/+ 4 months; (B) VaJ /VaJ 2 months. Melanosomes (arrow heads) clustered around the melanocyte nucleus underlie the basal lamina (bl) and dark cells (dk) of the crus commune. Scale bars = 1 Wm.
strial pigment and the presence of an EP in most of the mice studied. However, pigment distribution from many of the specimens shown in Table 2 was assessed by examination of sectioned cochleas so bands of pigmentation or reduced pigmentation may have gone unrecorded. This may explain the recording of a VaJ /VaJ mouse with an EP of 16 mV but no detected strial pigment. Flat strial surface preparations are the optimum way of recording the distribution of pigment along the entire length of the cochlea (see Fig. 7 in Cable et al., 1994), but we only made these preparations for 6 of the 19 mutants studied because we wanted to examine the whole cochlea in the remaining 13 mutants. Another VaJ /VaJ mutant (no. 41) with no EP had some pigment in the apical and basal turns, and a young VaJ / + (20 days old) with no EP appeared to have a normally pigmented stria. Even if part of this cochlea had reduced strial pigment, it is surprising that no EP was recorded. However, we did not section the entire cochlea of this specimen to check that the electrode was correctly positioned (e.g. Steel et al., 1987) and it is also possible that during EP measurement the tip of the electrode did not actually penetrate the scala media. The EP and distribution of strial pigment was only recorded from both ears in one animal. In this mature VaJ /VaJ mouse there was an asymmetrical pattern of
pigmentation : the unpigmented left ear had no EP whereas the right ear had reduced strial pigment and an EP of 45 mV. The distribution of melanocytes in the vestibular region of +/+, VaJ /+ and VaJ /VaJ is similar to that previously described in normally pigmented mice (Cable et al., 1994). There is no obvious ultrastructural di¡erence in vestibular melanocytes from controls and mutants (Fig. 5). Premelanosomes and melanosomes in the crus commune are slightly more spherical than those in the stria. The organ of Corti appeared normal in all control mice (Figs. 6A and 7A). Some degeneration of hair cells was present in all VaJ /+ mice but there was great variation in the number of abnormal cells and they tended to be scattered along the cochlea rather than restricted to speci¢c regions. Abnormalities of inner hair cells (IHC) were present in 14 day old VaJ /+ mice (the youngest animals examined in this study). Inner hair stereocilia were often fused together and reduced in number (Fig. 6C), or occasionally, completely absent. The cell cytoplasm was atypically electron-lucent and the apical surface of some IHC was swollen; the cuticular plate and surrounding belt of cytoplasm bulged out into the scala media (Fig. 6D). Fragmentation of these convex cuticular plates was common. In older mice,
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synaptic contacts at the base of the cells were rarely seen. Generally, outer hair cells (OHC) showed fewer signs of degeneration compared to the IHC with the
exception of one heterozygote (no. 16 that had no EP) in which many of the OHC had completely degenerated. Other OHC in VaJ /+ mice 18 days and older
Fig. 6. Inner hair cells in the middle cochlear turn from (A) +/+ 32 days; (B) VaJ /VaJ 36 days. Stereocilia (arrow) are missing from the mutant and the entire apical surface of the cell is swollen. (C) VaJ /+ 14 days, fused stereocilia (arrow head). Degenerating IHC, reduced in length with distorted apical surfaces, in (D) VaJ /+ 42 days; (E) VaJ /VaJ 18 days. Scale bars = 4 Wm.
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Fig. 7. Outer hair cells in the middle cochlear turn of (A) +/+ 18 days; (B) VaJ /VaJ 18 days; (C) VaJ /VaJ 20 days. Fused stereocilia (arrows), rounded vacuolated cells are characteristic of VaJ OHC degeneration. Scale bars = 4 Wm.
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had fused stereocilia. All supporting cells and Reissner's and tectorial membranes appeared una¡ected in the heterozygotes. Severe atrophy of the organ of Corti characterised the cochleas of VaJ /VaJ mice. IHC, when present, always had a swollen apical surface with an underlying thickened terminal web. Stereocilia were absent (Fig. 6B). Most IHC exhibited signs of degeneration (Fig. 6E) and contact with a¡erent nerve endings was rare. A few OHC retained their elongate shape with a basally positioned nucleus (Fig. 7B) but the majority consisted of condensed rounded cells with electron-lucent cytoplasm (Fig. 7C). Nuclei were intact often surrounded by lipofuscin granules but no e¡erent synapses were detected at the base of the cells. Stereocilia at the cell surface were fused together or absent. These IHC and OHC abnormalities present 14 days after birth became more widespread along the cochlea with age and were not restricted to any particular region of the cochlea. In mice 20 days and older, hair cells in some regions were completely atrophic. Elsewhere most OHC were shrunken, with just a few elongate cells remaining in the apical cochlea turn. IHC were even more sparsely distributed and their supporting cells were also degenerate. In all of the mice 53 days and younger (including those VaJ /VaJ mice with a reduced or absent EP), Reissner's membrane remained in its normal position. Similarly, no abnormalities of the tectorial membrane or spiral prominence were identi¢ed. In the oldest mouse examined (8 months old), atrophy of the hair and pillar cells was complete, just fragments of individual supporting cells remained. 4. Discussion The physiological and structural studies reported here suggest that the Varitint-waddler-J mutant has an unusual combination of primary defects in both the neuroepithelium and the stria vascularis. The stria defect is variable, and in extreme cases there is no recordable EP associated with an absence of melanocytes in the stria. We have observed a similar strial pathology in other mouse mutants with coat colour defects (Cable et al., 1992, 1994) and the ¢ndings strongly suggest that the lack of melanocytes in the stria causes strial dysfunction and a reduced EP. We do not know the reason for the lack of melanocytes, and it may be that the developing stria has a primary abnormality which directly a¡ects its ability to produce an EP as well as making it an inhospitable environment for survival of melanocytes. However, as we already know that melanocytes are essential for normal strial function, and that skin melanocytes are a¡ected by the VaJ mutation, a simpler explanation of a reduced EP is that the mutation causes the stria defect by its e¡ects on melanocytes.
It seems unlikely that the organ of Corti defects and the impaired cochlear responses in VaJ mutants are all a consequence of a reduced EP for several reasons. Firstly, in some mutants the EP (as recorded in the basal turn) was within the normal range but there was still a severe impairment in responses to sound. Secondly, in W and Sl mutants, which we have studied extensively, there is often no EP but hair cells survive and look normal for several weeks before they start to degenerate (Steel and Harvey, 1992 ; Steel et al., 1987); the hair cell defects seen in VaJ are more severe than this, suggesting that they may be a¡ected directly by the mutation. It is also unlikely that the strial defects are a consequence of a primary organ of Corti defect because in several neuroepithelial mutants we have studied, such as dn and je, there are severe hair cell abnormalities and a complete lack of cochlear responses, but the EP remains normal even in very old mice (Bock and Steel, 1983 ; Steel and Bock, 1983b). In some VaJ mice as young as 20 days, the stria is atrophic. There is no evidence to suggest that collapse of Reissner's membrane might be responsible for the reduced or absent EP in VaJ mice, although this characteristic has been reported as a secondary e¡ect of cochleo-saccular degeneration (e.g. Deol, 1970 ; Mair, 1973). For these reasons, we suggest that the VaJ mutation most likely affects both the stria and the organ of Corti directly. The lack of any detectable CAP in the mutants correlates with the lack of any behavioural response to sound. It suggests that little or no responses to sound are passed to the brain. However, the ¢nding of an SP in some of the homozygotes is interesting, because it suggests that, at least in some cases, the mutant hair cells can depolarise, and that the non-linear response of the hair cell to stimulation is preserved. The SPs observed in homozygotes are all negative, and in a control mouse negative SPs are thought most likely to originate from apical turn hair cell activity. Hence, this might indicate that hair cells in the apex might be responsible for the negative SP seen, correlating with the ¢nding that the apical turn organ of Corti is better preserved than the basal turn. However, in the pathological cochleas studied, and at the very high sound levels used to elicit the SP responses, it is probably not appropriate to assume that the responses recorded are generated at the same part of the cochlear duct as might be the case in normal mice. In the mutant mice, the frequency/place map and the balance between responses of di¡erent hair cell groups are likely to be abnormal. Ultrastructural studies of the VaJ cochlea revealed a progressive degeneration of the organ of Corti with age. However, hearing loss in these mice is already advanced 14 days after birth. At this age, the observed hair cell abnormalities (such as fused stereocilia, swollen cuticular plates) may not be su¤cient in themselves to account for the poor physiological responses. It is more
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likely that some biochemical alteration of the cells is responsible for hearing loss in young mice. In a previous study of the other allele of Varitint-waddler, Va, hair cells were studied in surface preparations following staining for succinic dehydrogenase activity (Steel, 1978). Control incubations suggested that the technique is relatively non-speci¢c and that substrates other than succinate may give a strong labelling reaction, and tissue permeability to the reagents may also a¡ect labelling (e.g. Steel and Bock, 1983b). Nonetheless, in normal cochleas the hair cells are strongly labelled compared with the weak labelling of the supporting cells. In a total of 55 Va/+ cochleas ranging from 10 to 100 days old, compared with 42 age-matched littermate controls, progressive hair cell degeneration was seen in the mutants. Furthermore, at the youngest stages, 10 and 15 days old, most of the hair cells were present but only a small proportion of them showed the normal strong labelling, scattered around the organ of Corti, while the rest were unlabelled. This suggests that there may be an early physiological abnormality of these hair cells preventing either their uptake or their reduction of the Nitro Blue Tetrazolium to form insoluble formazan, the basis of the labelling reaction. If a similar disruption in reagent uptake or dehydrogenase activity is present in VaJ mice this may explain the rapid degeneration of hair cells. The observed scattered loss of hair cells along the cochlea in VaJ mice 20 days and older is commonly observed in other mouse mutants such as dn and je (Steel, 1978; Steel and Bock, 1983b). This genetic abnormality is distinct from the localized patches of damage found in animals exposed to excess noise (e.g. Johnsson and Hawkins, 1976). Earlier work on the Va allele suggested that it is likely to be more severe than the VaJ allele in its e¡ect on the ear, because the coat colour defect is more extreme in Va and there are clear vestibular defects in Va but no obvious behavioural defects in VaJ (Cloudman and Bunker, 1945 ; Deol, 1954; Lane, 1972). It does appear from the current study that the pathology of the inner ear of VaJ is less extreme compared to that reported in Va mice. Steel and Harvey (1992) made a distinction between those neuroepithelial mutants with auditory responses (such as shaker-1) and those without (such as deafness); VaJ mice fall into the former category and Va into the latter. However, it is not possible from the published accounts to make detailed comparisons of the physiology of Va and VaJ ears. Mikaelian et al. (1965) found no responses at all in Va, but were not clear whether they were heterozygotes or homozygotes, what age they were, or the maximum SPL used. Berlin et al. (1969) found abnormal vocalisations in Va mice and suggested that this was associated with complete deafness in these mutants. However, again it is not clear whether Va/Va or Va/+ mice were used in this study. Detailed light microscope examination of Va
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cochleas was undertaken by Deol (1954) and Kocher (1960). They reported progressive degeneration of the stria from 3 weeks onwards in heterozygotes, and a particularly thin stria in homozygotes from an early stage. Degeneration of hair cells in Va mice was apparent from 11 days onwards and progressed rapidly with age. Hair cell loss was variable and often asymmetric but was more advanced than that observed by us in VaJ mice. For example, hair cell degeneration was reported to be complete in one 35 day old Va/Va mouse (Deol, 1954). Abnormalities of the tectorial membrane present in immature Va were not observed during the current ultrastructural study of VaJ mice. However, pigmentation defects are present in the cochleas of Va and VaJ . Deol (1954) and Kocher (1960) both reported strial defects in Va mice as early as 12 days after birth. The variable thickness of the stria reported in Va/Va mice (Deol, 1954) may be indicative of the spotting observed here in VaJ /VaJ mice. Deafness is often associated with pigmentation anomalies in humans and other mammals including mice, rats, mink, cats and dogs (e.g. Cable et al., 1994 ; Hageman and Delleman, 1977 ; Johnsson and Hawkins, 1976; Mair, 1973 ; Sugiura and Hilding, 1970 ; Tsujimura et al., 1991). In addition to the disrupted pigmentation patterns in the skin or coat, these animals exhibit spotting of internal organs. Spotting genes cause a lack of pigment cells rather than acting by disrupting melanin biosynthesis which occurs in the melanocytes of albinos. In spotting mutants, reduced strial pigmentation indicating a lack of melanocytes correlates precisely with reduced EP. Although little progress has been made over recent years in assessing the precise function of melanocytes within the stria (Schulte and Steel, 1994), there is an increasing awareness of the similarities of these cells in the skin and cochlea. Conlee et al. (1994) demonstrated that melanocytes in the stria undergo proliferation at a similar rate to those in the skin and suggested that the factors known to a¡ect replication and melanogenesis of skin pigment cells may also a¡ect those in the ear. Unilateral e¡ects are common in spotting, and so only one ear may be a¡ected (e.g. Cable et al., 1994; Bosher and Hallpike, 1965). In this study, both ears were examined in only one mutant but this example was su¤cient to demonstrate the presence of unilateral spotting in the cochleas of VaJ mice. A preliminary examination of the vestibular region revealed a normal distribution of melanocytes. However, insu¤cient specimens were examined to determine whether or not the spotting observed in the cochlea also occurred in other regions of the inner ear. In contrast to other spotting mutants previously examined, strial pigment in VaJ /VaJ mice is not always con¢ned to a single region of the cochlea but may be present in several pigmented patches. This distribution of strial pigmentation may
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be related to the unusual coat pattern of these mice. A combination of spotting and dilution in the coat of Va mice led Silvers (1979) to suggest a complex Va-locus. VaJ apparently arose from Va, which might suggest a partial reversion. Clearly, identi¢cation of this gene will shed important light on the genetics of pigmentation, in addition to revealing the molecular basis of this dual cochleo-saccular/neuroepithelial hearing loss. Acknowledgments We thank Mike Edel for technical assistance and N. Glenn for animal care. References Berlin, C.I., Majeau, D.A., Steiner, S., 1969. Hearing and vocal output in normal, deaf and intact mice. J. Aud. Res. 9, 318^331. Bock, G.R., Steel, K.P., 1983. Inner ear pathology in the deafness mutant mouse. Acta Otolaryngol. 96, 39^47. Bosher, S.K., Hallpike, C.S., 1965. Observations on the histological features, development and pathogenesis of the inner ear degeneration of the deaf white cat. Proc. R. Soc. London B 162, 147^170. Cable, J., Steel, K.P., 1991. Identi¢cation of two types of melanocyte within the stria vascularis of the mouse inner ear. Pigment Cell Res. 4, 87^101. Cable, J., Barkway, C., Steel, K.P., 1992. Characteristics of stria vascularis melanocytes of Viable Dominant Spotting (Wv /Wv ) mouse mutants. Hear. Res. 64, 6^20. Cable, J., Huszar, D., Jaenisch, R., Steel, K.P., 1994. E¡ects of mutations at the W locus (c-kit) on inner ear pigmentation and function in the mouse. Pigment Cell Res. 7, 17^32. Cloudman, A.M., Bunker, L.E., 1945. The varitint-waddler mouse. A dominant mutation in Mus musculus. J. Hered. 36, 259^263. Conlee, J.W., Gerity, L.C., Bennett, M.L., 1994. Ongoing proliferation of melanocytes in the stria vascularis of adult guinea pigs. Hear. Res. 79, 115^122. Cools, A.R., 1972a. Neurochemical correlates of the waltzing-shaker syndrome in the varitint-waddler mouse. Psychopharmacologia 24, 384^396. Cools, A.R., 1972b. Asymmetrical spotting and direction of circling in the varitint-waddler mouse. J. Hered. 63, 161^171. Dallos, P., 1986. Neurobiology of inner and outer hair cells: intracellular recordings. Hear. Res. 22, 185^198. Dallos, P., Schoeny, Z.G., Cheatham, M.A., 1972. Cochlear summating potentials: Descriptive aspects. Acta Otolaryngol. (Suppl.) 302, 1^46. Deol, M.S., 1954. The anomalies of the labyrinth of the mutants Varitint-Waddler, Shaker-2 and Jerker in the mouse. J. Genet. 52, 562^588.
Deol, M.S., 1970. The origin of the acoustic ganglion and e¡ects of the gene dominant spotting (Wv ) in the mouse. J. Embryol. Exp. Morphol. 23, 773^784. Hageman, M.J., Delleman, J.W., 1977. Heterogeneity in Waardenburg Syndrome. Am. J. Hum. Genet. 29, 468^485. Harvey, D., Steel, K.P., 1992. The development and interpretation of the summating potential response. Hear. Res. 61, 137^146. Johnsson, L.G., Hawkins, J.E., 1976. Degeneration patterns in human ears exposed to noise. Ann. Otol. Rhinol. Laryngol. 85, 725^739. Kocher, W., 1960. Untersuchungen zur genetik und pathologie der entwicklung von 8 labyrinthmutanten (Deaf-Waltzer-Shaker-mutanten) der maus (Mus musculus). Z. Verebungslehre 91, 114^140. Lane, P.W., 1972. Two new mutations in linkage group XVI of the house mouse, Flaky tail and Varitant-J. J. Hered. 63, 355-140. Mair, I.W.S., 1973. Hereditary deafness in the white cat. Acta Otolaryngol. (Suppl.) 314, 5^48. Mikaelian, D.O., Alford, B.R., Ruben, R.J., 1965. Cochlear potentials and VIII nerve action potentials in normal and genetically deaf mice. Ann. Otol. Rhinol. Laryngol. 74, 146^157. Schulte, B.A., Steel, K.P., 1994. Expression of alpha-subunit and beta-subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the WV or Sld loci. Hear. Res. 78, 65^76. Silvers, W.K., 1979. The Coat Colors of Mice: a Model for Gene Action and Interaction. Springer, New York, NY. Steel, K.P., 1978. Studies on Mice with Genetical and Experimentallyinduced Abnormalities of the Inner Ear. Ph.D. Thesis. University of London. Steel, K.P., 1991. Similarities between mice and humans with hereditary deafness. Ann. NY Acad. Sci. 630, 68^79. Steel, K.P., 1995. Inherited hearing defects in mice. Annu. Rev. Genet. 29, 675^701. Steel, K.P., Barkway, C., 1989. Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear. Development 107, 453^463. Steel, K.P., Brown, S.D.M., 1994. Genes and deafness. Trends Genet. 10, 428^435. Steel, K.P., Bock, G.R., 1983a. Hereditary inner-ear abnormalities in animals. Arch. Otolaryngol. 109, 22^29. Steel, K.P., Bock, G.R., 1983b. Cochlear dysfunction in the Jerker mouse. Behav. Neurosci. 97, 381^391. Steel, K.P., Harvey, D., 1992. Development of auditory function in mutant mice. In: Romand, R. (Ed.), Development of Auditory Systems 2. Elsevier, Amsterdam, pp. 221^242. Steel, K.P., Barkway, C., Bock, G.R., 1987. Strial dysfunction in mice with cochleo-saccular abnormalities. Hear. Res. 27, 11^26. Sugiura, A., Hilding, D.A., 1970. Cochleo-saccular degeneration in Hedlund white mink. Acta Otolaryngol. 69, 126^137. Tsujimura, T., Hirota, S., Nomura, S., Niwa, Y., Yamazaki, M., Tono, T., Morii, E., Hyung-Min, K., Kondo, K., Nishimune, Y., Kitamura, Y., 1991. Characterisation of Ws mutant allele of rats: A 12-base deletion in tyrosinase domain of c-kit gene. Blood 78, 1942^1946.
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