Age-related structural investigation of the Bronx waltzer mutant mouse cochlea: scanning and transmission electron microscopy

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Hearing Research, 13 (1984) 123-134 Elsevier

HRR 00459

Age-related structural investigation of the Bronx waltzer mutant mouse cochlea: scanning and transmission electron microscopy Marc Lenoir and RCmy Pujol INSERM

U.254, Laboratoire de Neurobiologie de I’Audition, CHR St -Charles, 34059 Montpellier Cedex, France (Received 2 August 1983; accepted 7 December 1983)

Cochleas of mice homozygous for the Bronx waltzer gene (symbol bu) were investigated using scanning (SEM) and transmission (TEM) electron microscopy. An age-related study was done from birth to postnatal day 100. With SEM, the arrangement of hair cells confirmed the unique feature of the bu/bo cochlea: the inner hair cells (IHCs) were either absent or abnormally haired but the outer hair cells (OHCs) appeared normal. No significant difference was observed with age. Using TEM, the remaining (20-25s) IHCs could be divided into two groups: normal-looking IHCs but with an abnormal synaptic pole, and abnormal, abortive-like IHCs. Very little if any sign of degeneration was observed whatever the age. OHCs displayed an almost normal cytology and pattern of innervation. The neurons of the spiral ganglion were very rare, even at birth. These findings suggest that the bo mutation should rather be classified in another group, than ‘degenerative’. The persistence of normal structures at OHC level is discussed in light of the cochlear physiology of the bu/bu: it again raises the question of the real role of OHCs in the peripheral auditory mechanisms. Bronx waltzer, hereditary deafness, cochlea, inner and outer hair cells

Introduction Since there are many similarities between inherited diseases of the inner ear in mouse and man, the mouse provides a good model for studying genetic malformations of the peripheral auditory system [3]. In addition, mouse mutants exhibiting specific structural abnormality of the cochlea could be of great interest in understanding the normal function of this structure. For example the Bronx waltzer mutation (symb. bu) in the mouse, a single autosomal gene mutation, has been considered, since its discovery by Deol and Gluecksohn-Waelsch [4], as a good model for approaching the role of the two kinds of cochlear receptors. Mice homozygous for the bu gene (bu / bu) exhibit the unique feature of having abnormal or absent inner hair cells (IHCs), whereas the outer hair cells (OHCs) apparently remain intact. Using light microscopy, Deol and GluecksohnWaelsch [4] and Deol [5] have reported that at birth a large proportion of IHCs were lacking or grossly abnormal. Only a few apparently normal IHCs could be found, which had a tendency to 0378-5955/84/$03.00

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disappear with age. The authors linked IHC defects to a malformation of pillars and a severe impairment of ganglion cells by considering the latter as a consequence of the former. In contrast, gross morphology of the organ of Corti appeared normal, as well as the OHCs, even in the oldest animals (8 months) they have studied. The hearing ability of the bo/ bu is severely impaired. Using the Preyer reflex, Deol and Gluecksohn-Waelsch [4] reported that some mice gave only slight responses to sound during a short period of time after the onset of hearing. A more precise physiological investigation [1,2] showed a total absence of the gross cochlear action potential (AP); the recording of cochlear microphonic (CM) was possible but it was greatly reduced; the only normal potential recorded was the negative summating potential (SP). While the absence of AP is related to the lack of IHCs, the small CM amplitude suggests that OHCs could also be pathologically affected by the mutation [2]. The limitations of light microscopic observations with regard to anatomo-functional relationships (see also recent studies in other mouse mutants [8,10]) prompted

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us to investigate the bv/bv cochlea using both scanning (SEM) and transmission (TEM) electron microscopes. The present study concerns the developmental aspects of the bv/bv cochlear histopathology. Material and Methods A total of 17 mice, homozygous for the bv gene were investigated. The following developmental stages were chosen: birth (n = 3) and postnatal days 9 (n = 4), 35 (n = 5) 60 (n = 2) and 100 (n = 3). The animals were bred in our laboratory from a stock maintained by Deol at the University College of London. The shaking or waltzing behavior of homozygous (bu/bu) mice was clearly distinguishable at birth. For TEM (n = 11) the cochleas were processed using a classical protocol (for details see [ll]). After decapitation, under anesthesia, the cochleas were quickly removed, and immersed for 1 h in 3.5% glutaraldehyde in phosphate buffer (0.2 M, pH 7.2). They were then post-fixed in osmium tetroxide (2% in buffer), dehydrated, and embedded in Spurr resin. Thin sections were cut from the different cochlear coils, mounted on Formvar-coated grids, stained, and examined with a Philips 300 electron microscope. For SEM the same fixative procedure was used, then the bony capsules of the cochleas were removed during dehydration in alcohol. The preparations were critical point dried in CO, and coated with gold. Observations were performed with a JEOL 25 S electron microscope. Results Scanning electron microscopy (SEM) With this technique, only co&leas of mice aged 9, 35 and 100 days were observed. On a first approach, after removing the tectorial membrane (Figs. l-3), no significant difference appeared in the specimens with respect to hair cell distribution. No quantitative data has been compiled on IHCs, but the proportion of remaining tufts of stereocilia did not vary with age and could be estimated at around 20-25% in all specimens studied. At the OHC level the cell distribution was normal, except for some supernumerary cells and/or a slight disorganization, more frequently found when IHCs

were missing and pillars absent or abnormal (Figs. l-3). The only difference between the three developmental stages concerned the surface appearance of the 9-day-old organ of Corti. characterized by the presence of numerous microvilli on the Kolliker and Deiter cells. A more detailed observation has been made on the pattern of stereocilia of both IHCs and OHCs. On IHCs, most of the tufts showed clear abnormalities such as missing stereocilia (Figs. 4, 5) or bent stereocilia (Fig. 6); prints of the IHC apical pole without a single stereocilium were also observed (Fig. 4). These malformations occurred in all specimens, studied even at day 9 (Figs. 1, 4); the only change with age was a more frequent occurrence of bent stereocilia in the oldest specimens (Figs. 3, 6). On OHCs, the tufts of stereocilia appeared perfectly developed and arranged at all stages (Figs. l-3). Again the only difference was linked to maturation: a kinocilium was present at day 9 (Fig. 4). The last finding concerning these surface SEM preparations is related to the arrangement of the pillar cell heads. A general disarray of these structures appeared, more frequently encountered when IHCs were missing (Fig. 2). As could be seen by a lateral approach after dissection of the inner pillars, this disarray involved the entire pillar and led to large and abnormal openings between adjacent pillars (Fig. 7). Transmission electron microscopy (TEM) At low magnification. it was possible to Birth. find a normal-looking organ of Corti in some sections (Fig. 8). Kiilliker’s organ was well developed, the hair cells were already differentiated and connected with nerve endings, the tunnel of Corti was not formed, but the pillar cells were distinguishable. Depending on the sections, great variation was seen in the IHCs. Beside some ‘normal’ IHCs (see Fig. S), many abnormal, abortivelike, IHCs were encountered (Fig. 9), displaying a small rounded shape, with no or greatly abnormal stereocilia. In other sections, no IHCs at all could be recognized and other structures (Kolliker cells on one side, pillars and OHCs on the other side) were closely packed, with no free space or cellular debris at the IHC position (Fig. 10). The pillar cells were sometimes absent or had abnormal cyto-

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Figs. 1-7. Scanning electron micrographs of the bv/bt~ mouse cochlea. Figs. l-3. Surface preparations of the organ of Corn, mice aged 9 days (Figs. 1, x 750), 35 days (Fig. 2. >: 1600) and 100 days (Fil g. 3, x 800). Except for the numerous microvilli seen at day 9, very few differences can be observed regarding the arrangement of hair cell stereocilia. The tufts of remaining inner hair cells (I) often show bent or missing stereocilia. The outer hair cell (0) stereocilia are always well preserved. Some supernumerary outer hair cells are found, more frequently when the facing irmer hair cell is missing. The pillar heads (P) are often disarranged and sometimes they lack completely (asterisk, Fig. 2).

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Fig. 4. Detail of the surface of the organ of Corti at day 9 (see also Fig. 1). The apical poles of Kiilliker (K) and Deiter (D) cells have numerous microvilli. Only two inner hair cells are clearly distinguishable on both sides of the photograp: h. At the base of tl re W pattern of outer hair cell stereocilia, the kinocilium can be seen (marked on two OHCs by arrowheads). x 3‘500. Figs. 5 and 6. Tufts of stereocilia on two remaining inner hair cells at day 35. Some stereocilia are missing (arrows, Fig. 5) or bent (Fig. 6). x 5000. Fig 7. Dissected portion of an organ of Corti at day 100. The inner pillars show abnormal arrangement wit1I large openings bet ween adjacent pillars. Note (arrows) the fibers (probably efferent) crossing the tunnel of Corti. X 2200.

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Figs. 8-12. Transmission electron micrographs of the bu/bu mouse cochlea at birth. Fig. 8. Low magnification of the organ of Corti. This section shows apparently well developed inner hair cell (I). The general shape of inner and outer (0) hair cells as well as the pattern of innervation are normal for this age. The Kolliker cells (K) are well developed. The tunnel of Corti is not formed. x 2800. Fig. 9. Section showing an abnormal, abortive-like, IHC (I). The pillar cells (P) have abnormal cytoplas mic contents. Outer hair cells (0) appear normal for this age (see Fig. 8). x 3000.

Fig. 10. Section showing no IHC, but some nerve fibers are trapped between Kiilliker (K) and pillar (P) cells. As in Fig. 9. the cytoplasm of pillar cells is abnormal, but the OHCs (0) look normal. X 4300. Fig. 11. Base of an outer hair cell (0). normally surrounded by afferent endings (a). A triple synaptic body (arrow) is seen opposite one afferent. X 10700. Fig. 12. View of the ganglion of Corti. Two neurons (N) are seen surrounded by many free spaces without degenerated cells or cellular debris. The glial cells (dark nucleus) ensheathe neurons and clusters of fibers. x 1800.

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Figs. 13-18. Transmission electron micrographs of the bu/bo mouse cochlea at postnatal day 10. Fig. 13. From the upper left to lower right, this picture shows Kolliker cells, an inner hair cell (I), pillar cells (P) and an outer hair cell (0) closely packed: sign of immaturity. X 2700. Fig. 14. Apical pole of an IHC. When the IHC is present (as in Fig. 13) it shows cytopiasmic signs of abnormalities such as lysosomes (arrows) and myehn figures (arrowhead). The retardation in development is indicated by the presence of a kinocihum (asterisk). x 8300. Fig. 15. Synaptic region at the base of an IHC (I). Afferent endings (a) have a flocculent cytoplasm. Contacts with the inner hair cell (arrows) are more desmosome-like than real afferent synapses. Efferents (e) make axo-dendritic synapses (arrowheads) with afferents. x 19 300. Fig. 16. Synaptic region of an outer hair cell (0). The cell is stih only surrounded by afferents which again indicates a retarded maturation. The cytoplasm of the OHC contains an abundance of smooth endoplasmic reticulum (arrows). x 12700. Fig. 17. Section through the ganglion of Corti showing two neurons (N), one myehnated, the other being only surrounded by the glial cytoplasm. The nerve fibers are well myelinated. No cellular debris is seen in the free spaces. x 1800. Fig. 18. High magnification of a ganglion neuron showing an abnormal cytoplasm with large patches of filamentous material and abundant smooth endoplasmic reticulum. X 14200.

Figs. 19-21. Electron micrographs from a cochlea of a 35-day-old bu/bu mouse. Fig. 19. Section showing an apparently normal IHC (I). The organ of Corti has completed its develc jpment, as indicated by the well formed tunnel (TC) and the disappearance of Kolliker cells on the IHC side. The neural elements lo ok normal below the IH C. Note the fibers (efferent) crossing the tunnel. X 3600. Fig. 20. Grossly normal OHC. Note the Nuel’s spaces on both sides and the normal reticular plate. x 4300. Fig. 21. Basal pole of an OHC showing both types of neural connections. The efferent is filled with nricrovesicles. Clusters of vesicles (arrowheads) are packed opposite the OHC, in which a postsynaptic cistern is seen. The afferent dendrite (a) penetrates into an invagination of the OHC membrane. X 8200. Fig. 22. lOO-day-old bo/bo mouse cochlea. Top of two OHCs (0) showing stereocilia and the points of anchorage (arrowhea Ids) into the tectorial membrane. The cuticular plate of both OHC and Deiter cells (D) looks normal. X 7300

plasmic contents (Figs. 9, 10). The OHCs had, in all sections, a normal appearance for this age with a rounded shape and a big nucleus; they were closely packed, and displayed a synaptic pole quite normal for this age, with exclusively afferent connections (Figs. 8-11). In the ganglion of Corti a severe lack of neurons was observed (Fig. 12). Here again, no cellular debris or degenerated cell was found where ganglion cells were missing. Day 10, The organ of Corti was still clearly immature: the Kijlliker cells still abundant, the tunnel of Corti not yet opened (Fig. 13) or just beginning to form. As described in the previous stage, the IHC appearance depended on the observed section: either ‘normal’ (very rarely: Fig. 13) or abnormal abortive-like, or no IHC at all. At this stage, some cytological details could be seen in the so-called ‘normal’ IHCs. Cytoplasmic abno~alities were found in these IHCs, such as vacuoles, lysosomes, myelin figures and an abundant smooth endoplasmic reticulum (Figs. 14, 15). At the synaptic pole, the junctions between the IHC and the afferent dendrites more closely resembled desmosomes or close appositions than normal afferent synapses. No presynaptic body or vesicle was encountered in the IHCs (Fig. 15). The afferent endings also displayed abnormal features with flocculent cytoplasm. In contrast, the efferents were well preserved and they made numerous axodendritic synapses (Fig. 15). At the OHC level (Fig. 16), the cell was still exclusively surrounded by afferents. The only cytoplasmic abnormality consisted in an abundant smooth endoplasmic reticulum. The ganglion neurons, even without precise quantitative analysis, appeared to be reduced to about 20%. There was still no sign of degenerated cells (Fig. 17). The rem~nin~ neurons displayed abnormal cytoplasmic contents such as filamentous material (Fig. 18). The myelination of intraganglionic fibers appeared normal (Fig. 17). Day 35. The organ of Corti now had a mature appearance, with an inner spiral sulcus, tunnel and Nuel’s spaces, with well developed OHCs and supporting cells, and normal-looking tectorial membrane. The same images as described in earlier stages were found for the IHCs and the proportion between normal, abnormal and missing IHCs, were generally the same. The remaining and apparently well developed IHCs may or may not be con-

nected with neural elements. When nerve endings were present (Fig. 19), both afferents and efferents were seen, but only atypical neuro-epithelial junctions were observed. Abnormalities, or even the absence of pillars were more easily detected at this stage, because of the formation of the tunnel. OHCs were fully developed at this age with regard to their shape and innervation pattern (Fig. 20). In particular, well formed efferent synapses were found at the basal pole of the OHCs (Fig. 21). The reticular plate seemed perfectly developed and intact, even when a pillar was missing. At the spiral ganglion level, some degenerative processes occurred in the remaining ganglion neurons. Very few changes were noted at Older stages. day 60, as compared with day 35. At day 100, more cytoplasmic abnormalities were found both in the remaining IHCs and the OHCs, but OHC stereocilia were still anchored in the tectorial membrane (Fig. 22). The cuticular plate presented a normal feature. Discussion When presenting the new Bronx waltzer mutation, Deol and Gluecksohn-Waelsch [4], using light microscopy, gave a fundamental description of its cochlear histopathology. They observed “a large proportion of IHCs not formed at all and of those that are formed the vast majority are grossly abnormal”. They also reported a severe and rapid degeneration of the ganglion of Corti, and a normal appearance for the OHCs. Naturally, they knew the limitations of the technique they used and called for further investigations using more sophisticated techniques. Both of these techniques (SEM and TEM) have been employed here and the results will be discussed mainly in the light of Deol and Gluecksohn-Waelsch’s assessments [4]. Inner hair cells Our SEM observations permitted a satisfactory evaluation of the number of IIICs present in bu/bu cochleas, as attested by their hairy apical pole. Grossly, the proportion (20-254) is comparable to that reported by Deol and Gluecksohn-Waelsch [4]. However, that proportion appears to be stable for the three stages studied (9, 35, 100 days). From stereocilia disorganization it can be supposed that

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many of these IHCs are not normal. With TEM, it was in fact almost impossible to find any completely normal IHC in all the specimens studied. The IHCs of grossly normal appearance in fact had a basal pole in which it was either impossible to find any neural connection, or these connections, especially between IHCs and afferents were hardly normal-looking synapses. The abnormal IHCs were generally dwarfish, or abortive-like, as if something abnormal had occurred at the time of cell differentiation. These abortive IHCs, generally without normal stereocilia, were seen in all the stages. Some of them possibly degenerate with age, but probably very few do so, as the signs of degeneration were rare, whatever the age of the animal. This must be emphasized, since Deol and Gluecksohn-Waelsch [4] favoured a degenerative process affecting IHCs. Concerning the missing IHCs, our observations support the hypothesis that the cells are missing from the very beginning: they are at least missing at birth and there is no sign of degeneration indicating a prenatal degeneration. Where IHCs are missing, Kolliker cells closely adjoin the pillar cells, with no empty space and/or IHC remnant in between. Regarding the supporting cells, our data agree with Deol’s findings [4,5] that pillars are the only affected structures. Some pillars were missing at birth, which again indicates an early problem with cytodifferentiation, and not a degenerative process following IHC impairment [4]. Moreover, there is some relationship between malformed or disorganized pillars and missing IHCs. Outer hair cells With SEM, the OHCs look quite healthy with regard to the arrangement of their stereocilia which are still perfectly normal at day 100. There is a slight tendency to row disorganization, with frequent supernumerary OHCs. The absence of IHCs and/or pillars may be responsible for this disarray. OHCs are in fact the last structure to achieve full development in the organ of Corti [7,9], and they may need a firm support to position correctly. TEM observations confirm, at least at low magnification, the normal appearance of the OHCs. Compared to normal mice [9], the bo/bv OHCs look the same at birth; they are slightly retarded in maturation at day 9 (still having a rounded shape);

at day 35 or 60 they appear normal; at day 100 some pictures reveal signs of degeneration: this may be related to aging, which seems to start very early in this species [ll]. At higher magnification, some weak cytoplasmic abnormalities could be found from day 10 on. But the synaptic pole of the cell is perfectly normal at day 35 and in older stages; in particular, the efferent innervation is normally developed and large efferent synapses are found, for which both the pre- and post-synaptic sides look normal (see [9]). Neurons of the ganglion of Corti The abnormalities described in this area mainly concern a lack of cells. At the youngest stage studied, i.e. at birth, most of the cells are missing and no sign of degeneration can be observed. This is in contradiction with Deol and GluecksohnWaelsch’s [4] findings that tended to link ganglion cell damage with IHC abnormalities. They referred to a rapid degeneration of neurons starting from an almost normal population at birth. Our results, on the contrary, tend to indicate an abnormal process of neuronal differentiation. as is the case with the IHCs and probably with the pillars. Apart from this lack of neurons, the ganglion cells which are present in ho/hr~ at very young stages have abnormal cytoplasmic contents such as vacuoles and fibrillar material. This has previously been observed in two other mouse mutants: the shaker-l [lo] and the deafness [8]. It has been related to a primary disorder in the neurons, due to a genetic effect at the embryonic stage of development. Classification of the bu / brl mutatmrl Considered as a whole, the results discussed above clearly demonstrate the impossibility of simply classifying bu/bu in the degenerative group [4,5,13]. The large majority of the organs of Corti in this mutant clearly do not degenerate, at least not during the first postnatal months. In br>/bu. the main problems for the structures involved in the mutation (IHCs, ganglion neurons, and maybe pillars), concern cytodifferentiation and/or proliferation. It even seems difficult to consider classifying this mutation in the intermediate group which has been proposed for shaker-l [lo] and deufness [8]. These mutants exhibit both problems,

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some linked to embryonic stages, and others to postnatal degeneration. In this respect, bu/bo is a very special mutation in which the organ of Corti, apart from the lack of IHCs, displays a normal appearance and maintains it for a long period. How can the difference be explained? The only possible hypothesis involves emphasizing the role played by the OHCs. Indeed, in the organs of Corti of bu/bu aged 35 or 60 days, the OHCs (and their efferent innervation) are the only neurosensory structures which have a normal appearance (except for some minute cytoplasmic abnormalities). These structures are perhaps necessary for maintaining the general aspect of the organ of Corti. OHCs normally anchored in the tectorial membrane, and a normal reticular plate at the OHC level are factors which may keep the organ of Corti in a normal fluid environment, preventing drastic degenerative processes. The situation is completely different in mutants, such as shaker-l or deafness, where OHCs are affected genetically and degenerate early together with the whole organ of Corti. Histo-physiological relationships Since its discovery [4] the bu/bu mouse mutant has been considered to be a useful tool in understanding the specific physiological properties of both types of cochlear receptors. Even the title of the paper (The role of IHC in hearing) is significant in this respect. The bu/bu cochlea was thought to be the only model in which only OHCs were normal. In fact, a detailed study of the bu/bu cochlear physiology [1,2] demonstrated that these mutants have no gross action potential (AP), a very low cochlear microphonic (CM), and a normal negative summating potential (SP). The absence of any normal IHC explains the absence of gross AP, but the finding of a very small CM (10% of the normal amplitude) is somewhat surprising. Indeed, the gross morphology of OHCs, their stereocilia, and the tectorial membrane is normal in bu/bu cochleas. As far as these structures are concerned, the cochlear micromechanics do not seem to be altered. If bu/bu OHCs are responsible for the abnormal CM, only certain metabolic problems indicated by the minor cytoplasmic abnormalities described above could be involved. However, explanations could also be proposed such

as an increased role of IHCs in producing CM [2], or a micromechanical impairment linked to IHCs and/or pillar absence or malformation. One of the main questions raised by the bu/bu cochlea is the normal appearance of the neurosensory connections between efferents and OHCs. This, together with the non-functioning stage (acoustically speaking) of the bu/bu cochlea, may infer that OHCs and their efferent innervations have physiological properties that are linked more to micromechanics [6,12] than merely to the sending of sensory messages. Acknowledgements This work has been supported by grants from INSERM (CRL 816022 and PRC 135027). The authors are indebted to Professor MS. Deol (University College of London) for his courtesy in providing the mice. They also gratefully acknowledge Mrs. S. Ladrech for her excellent technical assistance, and thank J. Goodfellow for editing work. References Bock, G.R. and Yates, G.K. (1982): Cochlear electrophysiology in the Bronx wolrrer mutant mouse. J. Physiol. (London) 332, 20-21. Bock, G.R., Yates, G.K. and Deal, MS. (1982): Cochlear potentials in the Bronx walrzer mutant mouse. Neurosci. Lett. 34, 19-25. Deol, MS. (1968): Inherited diseases of the inner ear in man in the light of studies on the mouse. J. Med. Genet. 5, 137-158. Deol, M.S. and Gluecksohn-Waelsch, S. (1979): The role of inner hair cells in hearing. Nature (London) 278, 250-252. Deol, M.S. (1981): The inner ear in Bronx waltzer mice. Acta Otolaryngol. 92, 331-336. Mountain, D.C. (1980): Changes in endolymphatic potential and crossed olivocochlear bundle stimulation alter cochlear mechanics. Science 210, 71-72. Pujol, R., Carlier, E. and Devigne, C. (1978): Different patterns of cochlear innervation during the development of the kitten. J. Comp. Neurol. 177, 529-536. Pujol, R., Shnerson, A., Lenoir, M. and Deol, M.S. (1983): Early degeneration of sensory and ganglion cells in the inner ear of mice with uncomplicated genetic deafness (dn). Hearing Res. 12, 57-63. 9 Shnerson, A., Devigne, C. and Pujol, R. (1982): Age-related changes in the C57B1/6J mouse cochlea. II. Ultrastructural findings. Dev. Brain Res. 2, 77-88.

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10 Shnerson, A., Lenoir, M., Van de Water, T.R. and Pujol, R. (1983): The pattern of sensory-neural degeneration in the cochlea of deaf shaker-l mouse: ultrastructural observations. Dev. Brain Res. 9, 305-315. 11 Shnerson, A. and Pujol, R. (1983): Development: Anatomy, electrophysiology and behavior. In: The Auditory Psychobiology of the Mouse, pp. 395-425. Editor: J.F. Willot. CC. Thomas, Springfield, Ill.

12 Siegel, J.H. and Kim, D.O. (1982): Efferent neural control of cochlear mechanics. Olive-cochlear bundle stimulation affects cochlear biomechanical nonlinearity. Hearing Res. 6. 171-182. 13 Steel, K.P., Niaussat, M.M. and Bock, G.R. (1983): The genetics of hearing. In: The Auditory Psychobiology of the Mouse, pp. 341-394. Editor: J.F. Willot. C.C. Thomas. Springfield, Ill.