The Inner Ear Macular Sensory Epithelia of the Daubenton's

THE JOURNAL OF COMPARATIVE NEUROLOGY 438:433–444 (2001)

The Inner Ear Macular Sensory Epithelia of the Daubenton’s Bat

METTE KIRKEGAARD AND JØRGEN MØRUP JØRGENSEN* Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACT The inner ear macular sensory epithelia of the Daubenton’s bat were examined quanti- tatively to estimate the area and total number of hair cells. Ultrastructural examination of the sensory epithelium reveals two main types of hair cells: the chalice-innervated hair cell and the bouton-innervated hair cell. The existence of an intermediate type, with a nerve ending covering the lateral side of the hair cell, indicates that the chalice-innervated hair cells are derived from bouton-innervated hair cells. Thus, at least a part of the bouton- innervated hair cells forms a transitional stage. A number of immature as well as apoptotic hair cells were observed. It is suggested that a continuous production of new hair cells takes place in mature individuals, probably based on transdifferentiation of supporting cells. J. Comp. Neurol. 438:433–444, 2001. © 2001 Wiley-Liss, Inc.

Indexing terms: hair cell; hair bundle; macula utriculi; transdifferentiation; development; turnover

Bats (order Chiroptera) are the only mammals that is a general mammalian feature is not known. The origin have developed active flight. It can be speculated that of the new hair cells is obscure because undamaged mam- mammals moving in three dimensions (i.e., flying or swim- malian vestibular epithelia show practically no cell divi- ming) could exhibit special conformations in the vestibu- sions (Jørgensen, 1991; Rubel et al., 1995; Kuntz and lar apparatus compared with their terrestrial counter- Oesterle, 1998). In this respect, bats are interesting ani- parts. Previous investigations have shown that the gross mals. They grow old compared with mammals of similar morphology of the chiropteran vestibular apparatus is size, and many of the Myotis species reach a maximum age typical mammalian (Gray, 1907; Iwata, 1924; Ram- of 20 years and have average life spans of 3–5 years prashad et al., 1980). However, according to the most (Schober and Grimmberger, 1987). Furthermore, they are recent investigation, the areas of the sensory maculae in dependent on vestibular function due to their mode of life. the little brown bat (Myotis lucifugus) are very small Thus, if any mammal should exhibit a continuous produc- compared with those of other mammals (Ramprashad et tion of hair cells in the vestibular organs, the bat is a likely al., 1980). Additionally, the density of hair cells in the candidate. macula utriculi was found to be extremely high, whereas In the present study, the maculae of the Daubenton’s the macula sacculi contained comparatively fewer hair bat (Myotis daubentonii, Kuhl 1819) were studied with cells. The density of hair cells in the maculae is consistent emphasis on the utricular macula. The study had two within most mammalian species (Lindeman, 1969; Lin- main objectives, the first of which was to investigate the denlaub et al., 1995), but the investigation by Ram- vestibular epithelia of a microchiropteran species in rela- prashad et al. (1980) indicates that the sensory epithelia tion to size, structure, and number of hair cells. Because of bats might differ from that of other mammals in this data on equal sized mammals are lacking, the vestibular respect. sensory epithelia of the common shrew (Sorex araneus, L.) Since the finding that ongoing hair cell formation exists were also investigated. The second aim was to look for any in normal avian vestibular sensory epithelia (Jørgensen and Mathiesen, 1988; Roberson et al., 1992), researchers have wondered whether the mammalian vestibular sensory epithelia may possess the same ability. Developing vestibular hair bundles have been observed in mature *Correspondence to: Jørgen Mørup Jørgensen, Department of Zoophysi- ology, Building 131, University of Aarhus, DK-8000 Aarhus C, Denmark. guinea pigs (Forge et al., 1993; Rubel et al., 1995; Lambert E-mail: [email protected] et al., 1997), but innervation was not confirmed. Whether Received 25 February 2000; Revised 18 May 2001; Accepted 25 June formation of new hair cells in vestibular sensory epithelia 2001

indications of turnover, i.e., developing and dying hair each sensory epithelium by using the estimated area and cells, in the macular epithelia of adult bats. the total hair cell number. The sensory epithelia used for scanning electron microscopy were ﬁxed as described above. Next the material was MATERIALS AND METHODS critical point dried, mounted on stubs, covered with plat- Ten specimens of Daubenton’s bat (M. daubentonii Kuhl inum in a sputtercoater, and examined in the scanning 1819) were collected at their winter quarters in October electron microscope (Maxim 2040, CamScan Electron Op- and February. After decapitation, the bony labyrinth was tics Ltd, Cambridge, U.K.) at 20 kV. By exposing the removed, opened, and ﬁxed in 3% glutaraldehyde in 0.15 material to ultrasound prior to the critical point drying, M phosphate buffer (pH 7.2) for 3 hours at room temper- the hair bundles were removed from the hair cell surfaces. ature. After a rinse in phosphate buffer, the material was The exact number of stereovilli was obtained by counting

postfixed in 1% OsO4 in phosphate buffer for 1 hour, the stubs at the apical cell surface. Counts were made on rinsed in phosphate buffer, and left in 70% ethanol over- 150 cell surfaces distributed over the pars externa, pars night. The next day the vestibular sensory areas were interna, and striola. The striola was recognized by the dissected in 70% ethanol and dehydrated in a graded orientation of the hair bundles, and countings were made series of ethanol. within two hair cells on each side of the dividing line. The Maculae utriculi from three left ears and one right ear total numbers of stereovilli per hair bundle as well as the were mounted on glass slides in XAM, neutral medium. number of rows from the kinocilium and the number of The right and left maculae sacculi from one specimen were stereovilli per row were counted. The averages for the pars likewise mounted on glass slides. The total number of hair externa, pars interna, and striola were compared by using cells was determined for each sensory epithelium by using the Kruskal-Wallis test for k-independent samples. Post a BX-50 Olympus microscope. Images were captured by hoc comparisons between areas were made due to signif- using a 3-CCD camera. Counting was performed with icantly different counts (Siegel and Castellan, 1988). CAST 2.0 software (Olympus Denmark A/S, Albertslund, The remaining material was embedded in Epon 812. For Denmark) by using the 2D-fractionator (Gundersen, transmission electron microscopy the epon blocks were sec- 1986). The 2D unbiased counting frame (Gundersen, 1977) tioned on an ultramicrotome (L. K. B. Ultrotome IV), picked had an area of 542.5 ␮m2 and was moved across the entire up on Formvar-coated copper grids, and stained with uranyl sensory epithelium in steps of 45 ␮m in both the horizon- acetate and lead citrate. One macula sacculi and one macula tal and the vertical directions. The total number of hair utriculi were cut tangential to the surface of the epithelium. cells is calculated as One macula sacculi and two maculae utriculi were cut perpendicular to the surface of the epithelium. The sections dxϫdy were examined in a transmission electron microscope (Zeiss N ϭ ϫ ͸ QϪ 10B, Zeiss Oberkochen, Germany). From two series cut per- A frame pendicular to the surface, the thicknesses of the stereovilli and the cuticular plate were measured for the two hair cell where dx and dy are the distance between counting frames types. Sixty stereovilli were measured from eight randomly ␮ in horizontal and vertical directions (45 m), Aframe is the chosen chalice-innervated hair cells. On average, eight ste- ␮ 2 Ϫ area of the counting frame (542.5 m ), and Q is reovilli were measured from each hair cell. From six ran- the number of hair cells counted in each counting frame. domly chosen bouton-innervated hair cells, 39 stereovilli The area was estimated by counting how many corners of were measured. On average, six stereovilli were measured the counting frame were found within the sensory epithe- from each hair cell. The thickness was measured as the lium at each sample point and calculated as diameter of the stereovillus at a height two-thirds of the total height. The measurements from chalice-innervated and dxϫdy bouton-innervated hair cells were compared as individual A ϭ ϫ ͸ P p observations by using a t-test. The thicknesses of cuticular plates from six randomly chosen chalice-innervated hair where p is the maximal number of corners (4) and P is the cells and seven randomly chosen bouton-innervated hair number of corners counted in each sample point. The cells were measured and compared by using a t-test. coefficient of error (CE) was calculated as For light microscopy, the blocks were cut into 2-␮m sections, mounted on glass slides, and stained with tolu- 1 idine blue. From tangential sections, the ratio between ϭ chalice-innervated and bouton-innervated hair cells was CE Ϫ ͱ͸ Q counted in two different maculae. The striola was defined as two hair cells on each side of the line dividing hair cells and found to be between 0.04 and 0.05 for all estimates with opposite orientation. Hair cells were counted within (Nyengaard, 1999). A mean number of 43 frames were an area of 50 ϫ 50 ␮m. Four samples were taken from the counted on maculae utriculi, and a mean number of 69 striola, and eight samples were taken from the extra frames were counted on maculae sacculi in Daubenton’s striolar areas. bat. The corresponding numbers for the common shrew To separate young animals from old ones, the degree of are 107 and 71, respectively. The average number of cells fusion of epiphyses in the distal part of the metacarpal of per frame was 10 in all specimens. The sensory epithelium the third finger was determined from X-ray radiographs. was discerned by the round apical surfaces of the hair The young, born in July, can by this method be discerned cells. Because the striola could not be discerned in the from adults until the following spring (Baagøe, 1977). All whole mount preparations, no separate counts were made animals except two exhibited complete fusion of epiphyses for the striola. The hair cell density was calculated for and were thus classified as adults. The remaining two THE MACULAR SENSORY EPITHELIA OF THE BAT 435

TABLE 1. Myotis: Data on the Maculae Utriculi and Sacculi Total Hair cell Weight Area hair cell density Specimen1 (g) (mm2) population (cells/mm2) macula utriculi 2L 12.31 0.071 1355 18,982 4L 10.21 0.049 982 19,747 5R 9.73 0.090 1545 17,149 6L 10.50 0.066 1347 20,475 Mean Ϯ SE 10.69 Ϯ 0.65 0.069 Ϯ 0.010 1307 Ϯ 136 19,098 Ϯ 829 macula sacculi 5R 9.73 0.146 2696 18,488 5L 0.144 2583 17,902 Mean Ϯ SE 0.145 Ϯ 0.001 2639 Ϯ 80 18,195 Ϯ 414

1R, right ear; L, left ear.

animals showed traces of epiphyseal closure, but because they were collected in February, they could be classified as “at least 6 months old.” For comparison, six specimens of the common shrew (Sorex araneus L.) were studied. The bony labyrinths were Fig. 1. Outline of the left macula utriculi of the Daubenton’s bat. fixed in 3% glutaraldehyde for between 3 and 24 hours but The orientation of the hair bundles is indicated with arrows. The were otherwise treated as previously described. The utric- arrow point indicates the position of the kinocilium relative to the ular maculae from five different animals were mounted on stereovilli. A, anterior direction; m, medial direction; pi, pars interna; glass slides in XAM, neutral media. Likewise, the saccular pe, pars externa; s, striola. Scale bar ϭ 100 ␮m. maculae from three different animals were mounted on glass slides. All animals were handled in accordance with NIH rules. TABLE 2. Sorex: Data on Maculae Utriculi and Sacculi Permission to collect and sacrifice bats was given by the Total hair cell Hair cell density National Forest and Nature Agency, Denmark. Specimen1 Area (mm2) population (cells/mm2) Macula utriculi 1R 0.220 RESULTS 2R 0.101 1810 17,880 3R 0.198 3233 16,289 Gross structure 3L 0.210 3986 18,975 Mean Ϯ SE 0.182 Ϯ 0.031 3010 Ϯ 781 17,715 Ϯ 955 The bony labyrinth of the Daubenton’s bat is isolated Macula Sacculi from the temporal bone by connective tissue. The mem- 3R 0.150 3L 0.129 2165 16,771 branous labyrinth contains five sensory organs in the ves- 6L 0.111 1885 16,925 tibular part and the organ of Corti in the cochlea. Each of Mean Ϯ SE 0.130 Ϯ 0.014 2025 Ϯ 198 16,848 Ϯ 109 the three semicircular ducts has an ampulla containing a 1R, right ear; L, left ear. crista. A transverse bar covered by nonsensory epithelia, the eminentiae cruciatae, divides the anterior and poste- rior cristae into two approximately equal parts. The three ampullae open into the utriculus, which contains the mac- lamina. At the luminal surface, they have numerous mi- ula utriculi. Below the utricular recessus is the sacculus, crovilli and a central ciliary rod (Fig. 2b). The supporting containing the macula sacculi. The macula sacculi is po- cells possess a reticular membrane seen as a dark band sitioned in a sagittal plane perpendicular to the macula below the apical surface (Fig. 2a,b). The chalice- utriculi. The maculae are overlaid by an otoconial mem- innervated hair cell (type I) is recognized by an afferent brane, which follows the outline of the sensory epithelium. nerve ending forming a chalice around the basolateral Quantitative data for the maculae of the Daubenton’s part of the cell (Figs. 2a, 4c). A constriction below the bat are given in Table 1.The area of the macula utriculi cuticular plate forms a neck and gives the cell a charac- was found to vary between 0.049 mm2 and 0.090 mm2, teristic flask-shaped appearance. In the apical region, nu- with a mean of 0.069 Ϯ 0.01 mm2 (mean Ϯ SE; n ϭ 4). The merous mitochondria are seen. The bouton-innervated mean total number of sensory cells in the macula was hair cell (type II) is ovoid to columnar in shape. It is 1307 Ϯ 136 (n ϭ 4). The outline of a representative utric- innervated by afferent and efferent nerve endings forming ular macula is given in Figure 1. The mean area of the boutons at the basal parts of the cell (Figs. 2a, 4c). maculae sacculi from one animal was found to be 0.145 Ϯ In the cytoplasm, numerous mitochondria and vesicles 0.001 mm2 (n ϭ 2), and the mean total number of cells was are seen. Both cell types exhibit synaptic structures op- 2639 Ϯ 80 (n ϭ 2). For comparison, the corresponding data posed to the nerve endings. These synaptic bodies occur for the common shrew are given in Table 2. individually or in pairs and may be spherical or elongated bars. The nuclei of the chalice-innervated hair cells are Structure of the sensory epithelium located in the lower part of the epithelium, above the The macular sensory epithelium contains supporting supporting cell nuclei, whereas the nuclei of the bouton- cells, nerve fibers, and two types of sensory cells. The innervated hair cells are generally located more luminally supporting cells span the height of the entire epithelium, (Fig. 2a). Both types of hair cells have an apical hair and their irregular nuclei are situated close to the basal bundle consisting of an eccentrically located kinocilium Fig. 2. Transmission electron micrographs of the epithelium of membrane (rm) of the supporting cells is seen as a dark band below macula utriculi showing the two types of hair cells. a: A chalice- the epithelial surface. b: The hair bundles of a bouton-innervated (B) innervated hair cell and a bouton-innervated hair cell. The bouton- and a chalice-innervated (C) hair cell. The latter has thick stereovilli innervated hair cell is contacted by a large nerve ending (n) covering with flat tips and a dense cuticular plate. One supporting cell dem- the lateral side of the cell and a conventional bouton (*). The chalice- onstrates the ciliary rod (arrow). c: The hair bundle of a chalice- innervated hair cell is recognized by a narrow neck and a nerve innervated hair cell. The stereovilli have flat tips and anchors with chalice (nc) surrounding the basolateral part of the cell. The reticular fine rootlets in the cuticular plate (arrow). Scale bars ϭ 2 ␮m. THE MACULAR SENSORY EPITHELIA OF THE BAT 437 and a number of stereovilli arranged in rows of decreasing However, there was no quantitative investigation of the height from the kinocilium. Each stereovillus anchors occurrence of apoptotic cells due to incomplete series. with a fine rootlet in the cuticular plate (Fig. 2c). However, there are differences between the hair bundles of the two Size and shape of hair bundles cell types. The stereovilli of the chalice-innervated hair The number of stereovilli per hair bundle varies within cells are thicker than the stereovilli of the bouton- the sensory epithelium. In the striola, the mean number of innervated hair cells, and they have flat tips. The stere- stereovilli per hair bundle is significantly lower compared ovilli of the chalice-innervated hair cells were estimated to with the extrastriolar areas (35.4 Ϯ 0.9 (n ϭ 52) vs. 53.7 Ϯ have a diameter of 0.168 Ϯ 0.004 ␮m(nϭ 60), and the 1.4 (n ϭ 89); p Ͻ 0.01). This difference is due to fewer rows stereovilli of the bouton-innervated hair cells were esti- of stereovilli in the striolar hair bundles (3.8 Ϯ 0.13 vs. mated to have a diameter of 0.128 Ϯ 0.003 ␮m(nϭ 39), 6.3 Ϯ 0.16; p Ͻ 0.01), whereas the number of stereovilli but this difference was not found to be significant. Fur- per row is the same in the striola and the extrastriolar thermore, the cuticular plate of the chalice-innervated areas (10.0 Ϯ 0.19). The limited number of rows gives the hair cells appears thicker and denser than the cuticular striolar hair bundles a distinctive flattened appearance plate of the bouton-innervated hair cell, but a statistically (compare Fig. 3a and b). From tangential sections through significant difference in the thickness of the cuticular the epithelium, it is evident that the flat hair bundles in plate could not be confirmed (0.783 Ϯ 0.085 ␮m vs. 0.689 Ϯ the striola belong to chalice-innervated hair cells, whereas 0.143 ␮m[nϭ 6, n ϭ 7]; Fig. 2b). the chalice-innervated hair cells in the extrastriolar areas The bouton-innervated hair cells vary from a spherical have normal looking hair bundles. to a cylindrical, almost flask-shaped type of cell, resem- bling the chalice-innervated cells. In addition to the afferent boutons innervating the hair cells basally, there are DISCUSSION laterally situated synapses on both bouton- and chalice- innervated hair cells. Some of the bouton-innervated hair Size of the sensory epithelia cells are innervated by large flat boutons that cover a The area of the macula utriculi of the Daubenton’s bat substantial part of the lateral side of the cell (Fig. 2a). was estimated to be 0.069 mm2 and the area of the macula The striola, recognized by the orientation of the hair sacculi to be 0.145 mm2. The estimate for the saccular bundles, runs parallel to the anterolateral margins and epithelium should be viewed with reservations, because it divides the sensory epithelium into a medial pars interna is based on the right and left sensory epithelia from one and a lateral pars externa (Fig. 1). The striola is domi- animal. In the study by Ramprashad et al. (1980), the nated by chalice-innervated hair cells, the chalice type/ corresponding areas in the little brown bat (M. lucifugus) bouton type ratio being 3.9:1. In the extrastriolar areas, were estimated to be 0.016 mm2 and 0.098 mm2, respec- the same ratio is 1.3:1. In the striola, the chalice- tively. These results agree with the present study in two innervated hair cells occur in chalices enclosing one, two, ways: the macular sensory epithelia are very small, and or three cells, whereas the extrastriolar region mainly the saccular macula is considerably larger than the utric- contains chalices with a single hair cell. ular macula. However, estimates of the macular areas and Supposed immature hair bundles were observed in all the total hair cell population differ in the two bat species, utricular and saccular maculae investigated. In tangential which are of equal size—a fact that may be due to the sections they are recognized by a hair bundle consisting of different methods used. The present study concludes that a central kinocilium surrounded by thin stereovilli (Fig. the vestibular apparatus of the Daubenton’s bat is typi- 3). Eleven immature hair bundles were found in a single cally mammalian and that the extreme relations reported utricular macula, but due to incomplete series, the total in the sensory epithelia of the little brown bat do not number per macula could not be obtained. In the remain- pertain to the Daubenton’s bat. ing maculae, between two and five immature hair bundles When comparing the area of the maculae in the were observed. Most of the immature hair bundles were Daubenton’s bat with the common shrew, an animal of found close to the striola and occurred in clusters (Fig. 3). equal size, the maculae utriculi of the common shrew In sections cut perpendicular to the surface of the sensory proved to be more than twice the area of the corresponding epithelium, the position of the kinocilium was difficult to sensory epithelium in the Daubenton’s bat, whereas the locate. However, some of the bouton-innervated hair cells saccular sensory epithelia were of equal size (Tables 1, 2). differed from the type described above. The cell body To investigate whether this difference is due to a deviation spans a large part of the epithelium, but no contact with in one or the other of the two species, they were compared the basal lamina was observed. The cuticular plate is with available data for other mammals. Data on guinea indistinct or absent, and mitochondria are visible in the pig (Lindeman, 1969), two mole rat species, and rat (Lin- supranuclear region. Some of these hair cells have an denlaub et al., 1995) are shown together with results from irregular/oval nucleus located basally whereas others the present study (bat and shrew) in Table 3. All the data have the nucleus situated in the central region of the cell included rely on whole mount preparations. The large body. They are innervated basally with bouton nerve end- relative size of the sensory epithelia in bat and shrew may ings. These hair cell bodies are considered to belong to the reflect their small size. One might expect the area of the immature hair bundles (Fig. 4c). sensory epithelia to be roughly correlated with size (i.e., In ultrathin sections from all four maculae investigated, weight). However, if minimum numbers of sensory recep- apoptotic cells were observed in the sensory epithelium. tors are required for adequate sensitivity/resolution, there They have a dark staining (pyknotic) nucleus, and the cell may be a lower limit to the total number of hair cells, plasma is vesiculated and condensed. Apoptosis was ex- which implies a minimum size of the epithelia. This would clusively seen in single chalice-innervated hair cells or in result in small animals exhibiting relatively larger sen- hair cells in chalices shared with normal cells (Fig. 4a,b). sory epithelia. Due to the limited amount of available data Fig. 3. Transmission electron micrographs of a tangential section through the striola. The hair bundles have fewer rows of stereovilli of hair bundles in the macula utriculi. a: The extrastriolar area, pars (compare with hair bundles in a). Two of the hair bundles have a externa. Most hair bundles are oriented toward the striola, i.e., the central kinocilium and are thus classified as immature (arrows). Ar- kinocilium is located peripherally. Three immature hair bundles (arrows in lower right corner indicate orientation of both sections: A, rows) have thin and unorganized stereovilli compared with the ma- anterior direction on the macula; M, medial direction on the macula. ture hair bundles and a central kinocilium. b: Tangential section Scale bars ϭ 2 ␮m. Fig. 4. a: Nerve chalice with an apoptotic cell co-occurring with a normal cell. The nucleus is pycnotic and the cytoplasm is vesiculated. b: Chalice-innervated apoptotic cell. c: Two chalice-innervated hair cells and an immature hair cell. The latter has an oval nucleus and is contacted by two boutons (arrows). Scale bars ϭ 2 ␮m. 440 M. KIRKEGAARD AND J.M. JØRGENSEN

TABLE 3. Data on Maculae Utriculi and Sacculi From Different ratio between the macular areas lies either close to or Mammalian Species above the line y ϭ x, i.e., the macular sensory epithelia are Total hair Hair cell of equal size or the utricular macula is larger than the Weight Area Relative cell density saccular macula. Because bats are the only flying mam- 2 1 2 Species (g) (mm ) area population (cells/mm ) mals, a possible explanation could be that the large sac- Macula utriculi cular macula reflects an adaptation to flight. It could be Myotis 11 0.069 0.629 1307 18,885 Sorex 12 0.182 1.520 3010 16,503 supposed that bats are subjected to vertical acceleration Cryptomys 80 0.470 0.588 8100 17,234 more frequently than ground-living mammals and hence Spalax 120 0.509 0.424 8470 16,640 have a well-developed vertical macula. If the deviation in Rattus 220 0.360 0.164 6020 16,722 Cavia 300 0.541 0.180 9260 17,116 the chiropteran vestibular epithelia is a mammalian ad- Macula sacculi aptation to the aerial mode of life, it would be obvious to Myotis 11 0.145 1.319 2639 18,197 Sorex 12 0.130 1.083 2025 15,577 compare bats with marine mammals or primates with an Cryptomys 80 0.429 0.536 7040 16,410 arboreal lifestyle. Like flying animals, animals swimming Spalax 120 0.483 0.403 7400 15,321 Rattus 220 0.381 0.173 5960 15,643 in water are expected to move equally in three dimen- Cavia 300 0.495 0.165 7560 15,273 sions, as opposed to terrestrial animals, which, in spite of jumping/climbing, are mainly subjected to horizontal ac- 1Relative area ([area/weight] ϫ 100). Data on Myotis and Sorex are from the present study. Data on Cryptomys, Spalax, and Rattus are from Lindenlaub et al. (1995). Data celerations. Likewise, mammals living in trees are fre- on Cavia are from Lindeman (1969). quently subjected to vertical accelerations. However, in the squirrel monkey, the utricular and saccular maculae are of equal size (Igarashi et al., 1975), and there are no investigations of the exact dimensions of the sensory epithelia in marine mammals. It must be concluded that so far there is no clear explanation of why the saccular macula is larger than the utricular macula in bats. Structure of the sensory epithelium The macular sensory epithelia of the Daubenton’s bat exhibit general mammalian features: bouton- and chalice- innervated hair cells surrounded by supporting cells and a striola in the utricular macula that is dominated by chalice-innervated hair cells. A striolar zone, dominated by chalice-innervated cells, is known from other mammals, including guinea pig (Lindeman, 1969; Watanuki and Meyer zum Gottesberge, 1971; Watanuki et al., 1971), echidna (Jørgensen and Locket, 1995), and human (Rosen- hall, 1972). The ratio between chalice-innervated and bouton-innervated hair cells found in the striola of guinea pig is approximately 2:1, whereas the extrastriolar area has a ratio of 1:1 (Lindeman, 1969; Watanuki and Meyer zum Gottesberge, 1971; Watanuki et al., 1971). In the present study the striola is found to be almost exclusively populated by chalice-innervated hair cells (ratio of chalice- innervated/bouton-innervated cells ϭ 3.9:1). Data from Fig. 5. The area of macula utriculi plotted against the area of other mammalian maculae are lacking, but the ratio of 2 ϭ macula sacculi (mm ). The macular ratios are close to the line y x chalice-innervated/bouton-innervated hair cells is known except for the two bat species, which have large saccular maculas relative to the utricular maculas. Solid square, little brown bat (Myo- to vary in the mammalian cristae (3:1 in squirrel monkey; tis lucifugus; data from Ramprashad et al., 1980); open square, 1:1 in chinchilla; Goldberg et al., 1992). The number of Daubenton’s bat (Myotis daubentonii; data from the present study); cells per chalice is lower in mammals than in the other solid diamond, common shrew (Sorex araneus; data from the present amniotes. In the guinea pig, two to three hair cells can be study); open diamond, rat (Rattus norvegicus; data from Lindenlaub found in the same chalice in the striola (Watanuki and et al., 1995); solid triangle, Zambian common mole rat (Cryptomys sp.; data from Lindenlaub et al., 1995); open triangle, blind mole rat Meyer zum Gottesberge, 1971; Watanuki et al., 1971), (Spalax ehrenbergi; data from Lindenlaub et al., 1995); solid circle, which corresponds well to the findings of the present study guinea pig (Cavia sp.; data from Lindeman, 1969). in which some chalices were found to contain two or three cells. In birds, the chalices contain up to 10 cells per chalice (Jørgensen, 1989), whereas the reptilian chalices enclose 1–5 cells, 1–3 being the most common (Jørgensen, on other mammals, it is difficult to deduce any clear rela- 1988). tions between the area of the vestibular sensory epithelia Lindeman (1969) and Lindeman et al. (1973) have pre- and animal size. viously noted the flat hair bundles in the striola of the In the Daubenton’s bat and in the little brown bat (Ram- utricular macula in guinea pig. They assumed that the prashad et al., 1980), the saccular macula is considerably hair bundles belong to chalice-innervated hair cells, due to larger than the utricular macula. This feature is illus- a large free surface area and club-shaped stereovilli. In trated in Figure 5. In the graph, the area of the macula the present study, the hair cells with flattened hair bun- utriculi is plotted against the area of the macula sacculi. dles are followed through the epithelium in serial sections. This reveals that in all the other mammalian species, the These tangential sections verify that the flat hair bundles THE MACULAR SENSORY EPITHELIA OF THE BAT 441 belong to chalice-innervated hair cells. Furthermore, the processes to compensate for the hair cells lost by apopto- difference in shape between hair bundles in the striola sis. and the extrastriola is quantified. It has been shown that the hair bundles in the striola have significantly fewer Immature hair cells in mature animals stereovilli and that this is due to significantly fewer rows The bouton-innervated hair cells with a central kinoci- of stereovilli. This arrangement of the stereovilli gives the lium, observed in the present study, are interpreted as striolar hair bundles a resemblance to the inner hair cells developing hair cells. This is based on several descriptions in the organ of Corti. In the present study, the total of the ontogenetic development of the vestibular sensory number of stereovilli per hair bundle was found to be 35 in epithelia in mouse and chick (Mbiene et al., 1984; Taku- the striola and 54 in the extrastriolar areas. This is low mida and Harada, 1984; Dechesne et al., 1986; Tilney et compared with previous investigations, which report be- al., 1992a,b) and descriptions from the vestibular sensory tween 50 and 100 stereovilli in guinea pig vestibular hair epithelia in guinea pigs, regenerating after ototoxic dam- bundles (Lindeman, 1969) and between 60 and 100 stere- age (Forge et al., 1993, 1998; Rubel et al., 1995; Li and ovilli in the squirrel monkey (Spoendlin, 1965). The func- Forge, 1997). These studies show that in the early stage of tional significance of these differences is unknown. hair bundle formation, numerous stereovilli of equal In the present study, the chalice-innervated hair cell is height form around a central kinocilium on the apical found to possess thicker stereovilli and a more voluminous surface of the hair cell. Later the kinocilium is found close cuticular plate than the bouton-innervated hair cell, even to the periphery, and the stereovilli attain decreasing if these differences are not statistically significant. Simi- height with increased distance from the kinocilium. Next, lar observations have been noticed in other amniotes (Ly- the stereovilli widen by adding more actin filaments. Fi- sakowski, 1996). This can be explained by the fact that the nally, a cuticular plate develops, and the stereovilli elon- chalice-innervated hair cells are ontogenetically older gate to the final height. This course of events corresponds than the bouton-innervated hair cells (see below). well to the types of hair bundles observed in the present study. Apoptotic cells in normal epithelia From a previous study (Kirkegaard and Jørgensen, 2000), it is evident that when followed through the epi- Apoptotic cells are observed in the macular epithelia of thelium in a series of sections, the immature hair bundles the Daubenton’s bat. Apoptosis is a type of programmed prove to belong to bouton-innervated hair cells. These cell death that occurs during tissue development and as immature, bouton-innervated hair cells are believed to part of the turnover in normal tissue (Wyllie, 1981; Alison correspond to the subtype of bouton-innervated hair cells and Sarraf, 1992; Jacobson et al., 1997). A low degree of observed in sections cut perpendicular to the surface of the apoptosis is observed in normal mammalian vestibular sensory epithelia. These hair cells have no visible cuticu- sensory epithelia (Li et al., 1995; Kuntz and Oesterle, lar plate and are thus regarded as an early stage in the 1998; Zheng et al., 1999). In the avian vestibular sensory development. epithelia, the continuous production of hair cells (Jør- Spoendlin (1965) observed hair bundles with a central gensen and Mathiesen, 1988; Roberson et al., 1992; kinocilium in the vestibular epithelia of the squirrel mon- Weisleder and Rubel, 1992; Tsue et al., 1994; Weisleder et key but ascribed it to the irregular orientation in some al., 1995) is probably regulated by apoptotic cell death, to hair bundles. Recently, similar hair bundles have been maintain a constant number of hair cells (Jørgensen, observed in mature guinea pigs (Forge et al., 1993; Rubel 1991; Roberson et al., 1992; Kil et al., 1997). Additionally, et al., 1995; Lambert et al., 1997; Forge et al., 1998). The apoptosis is recognized as a mode of hair cell degeneration present study confirms the existence of hair cells with an following aminoglycoside insult (Li et al., 1995, 1997; immature morphology in a mature mammal and further Lang and Liu, 1997; Nakagawa et al., 1998; Forge et al., that the immature hair cells are innervated with afferent 1998; Zheng et al., 1999). The apoptotic cells observed in synapses, which indicates normal function. We do not the present study are sparse and found scattered among know whether these hair cells differentiate any further. healthy cells. This indicates that they are not the result of Alternatively, they could represent a subgroup of hair chemical or mechanical damage during preparation, in cells not previously described. accordance with a recent examination of the fish inner ear (Jensen and Jørgensen, 2001). Unfortunately, it was not Origin of immature hair cells possible to quantify the apoptotic cells with the present If the hair cells with immature morphology are devel- material. oping hair cells, their origin is obscure. The avian vestib- Only chalice-innervated hair cells are recognized as ap- ular sensory epithelia are capable of continuous produc- optotic. This may be a coincidence, because observations of tion of hair cells (Jørgensen and Mathiesen, 1988; apoptotic hair cells are very sparse. However, this obser- Roberson et al., 1992; Tsue et al., 1994; Weisleder et al., vation supports the hypothesis of a continuous develop- 1995), but previous experiments with [3H]thymidine la- ment from the bouton-innervated hair cell to the chalice- beling in mammalian vestibular epithelia show no prolif- innervated hair cell (see below). Because the chalice- eration (Jørgensen, 1991; Rubel et al., 1995; Kuntz and innervated hair cells are ontogenetically the oldest, they Oesterle, 1998). Hence, it is doubtful whether the imma- may be expected to be more frequently subjected to apo- ture hair cells observed in the present study are the re- ptosis. The total number of hair cells declines with age in sults of immediately preceding mitoses. the vestibular epithelia in humans (Rosenhall, 1973) and One possibility is that partially injured hair cells are re- mice (Park et al., 1987). It is not known whether this is the growing a new hair bundle. It has been demonstrated that case in the vestibular epithelia of bats. However, it is after sublethal damage (mechanical or ototoxic) in which the likely that a long-lived animal such as the bat, depending hair cell loses its hair bundle, it will relocate the kinocilium on vestibular function, should exhibit some regenerative to the center of the cell surface and regrow a new hair bundle 442 M. KIRKEGAARD AND J.M. JØRGENSEN

Fig. 6. Schematic outline of the suggested development of the established, the hair cell is recognized as a bouton-innervated hair mammalian vestibular hair cell. A normal supporting cell with irreg- cell (3). Some of the bouton-innervated hair cells are contacted by ular nucleus close to the basal lamina (1). The supporting cell loses nerve endings, which enlarge and form a chalice (4). A further in- contact with the basal lamina and becomes innervated by nerve crease in the number of actin ﬁlaments results in thicker stereovilli endings. At the apical surface, stereovilli start to form around a and a denser cuticular plate. The ﬁnal stage is a chalice-innervated central kinocilium and the supporting cell is now considered an im- hair cell (5A). If the hair cell is situated in the striola, the hair bundle mature hair cell (2). When the hair bundle is formed and synapses is reduced (5B). L, luminal surface; B, basal lamina.

(Sobkowicz et al., 1996, 1997; Zheng et al., 1999). This will mammalian sensory epithelia, it might well be a way of result in hair bundles with a morphology similar to the one renewing hair cells in a continuous turnover. Of course, observed in the present study. However, the elongated cell the production of new hair cells by transdifferentiation of body of the observed immature hair cells and the basally supporting cells will eventually deplete the supporting cell located nucleus imply that the deviation from mature hair population. The population of supporting cells could be cells is not limited to the hair bundle. renewed by short periods of mitotic activity. This would An alternative explanation is that the developing hair explain the difficulties in labeling mitoses in undamaged cells originate from supporting cells through transdiffer- mammalian sensory epithelia. entiation, which is the phenotypic conversion of a cell at a late developmental stage (Beresford, 1990; Eguchi and Hair cell types Kodoma, 1993). Transdifferentiation has previously been In the present study, intermediate types between the suggested in regenerating inner ear epithelia of the bull- chalice- and bouton-innervated hair cell were observed. frog (Baird et al., 1996) and the chick (Weisleder et al., The cells innervated by boutons as well as by a large nerve 1995). In the regenerating mammalian epithelia, transdif- ending covering half of the cell (Fig. 2a) resemble descrip- ferentiation has been considered, because the number of tions from the ontogenetic development in the vestibular new hair cells observed in SEM disagrees with the num- sensory epithelia of cat and mouse (Favre and Sans, 1979; ber of dividing cells observed in the epithelium after oto- Nordemar, 1983). From these studies, it is evident that toxic damage (Warchol et al., 1993; Rubel et al., 1995; Li the developing chalice-innervated hair cell passes through and Forge, 1997). Studies by Li and Forge (1997) and a stage where it resembles the bouton-innervated hair Forge et al. (1998) report morphological indications of cell. Nerve endings covering the lateral part of a bouton- transdifferentiation in the regenerating vestibular epithe- innervated hair cell have previously been observed in ma- lia of the guinea pig. Cells with supporting cell features ture animals (Engstro¨m, 1961; Bagger-Sjo¨ba¨ck and Gul- such as a reticular membrane and contact with the basal ley, 1979) and have been suggested to be a developmental lamina exhibited immature hair bundles. stage between the two types of hair cells (Engstro¨m, In the present study, several morphological traits indi- 1961). Finally, the chalice-innervated hair cells have cate transdifferentiation. Like the supporting cells, the thicker stereovilli and a denser cuticular plate, which immature hair cells span a large part of the epithelium. indicate a later developmental stage (cf. the above descrip- The nuclei are irregular, and some cells have the nucleus tion of the ontogenetic development of the hair bundle). situated basally, close to the supporting cell nuclei. It is Together, these data imply that at least some of the suggested that because transdifferentiation of supporting bouton-innervated hair cells are merely an early develop- cells seems to be part of the regeneration in damaged mental stage of the chalice-innervated hair cells. A con- THE MACULAR SENSORY EPITHELIA OF THE BAT 443 tinuous development from a bouton-innervated hair cell to Forge A, Li L, Corwin JT, Nevill G. 1993. Ultrastructural evidence for hair a chalice-innervated hair cell has previously been de- cell regeneration in mammalian inner ear. Science 259:1616–1619. scribed in the regenerating vestibular epithelia of the Forge A, Li L, Nevill G. 1998. Hair cell recovery in the vestibular sensory chick (Weisleder et al., 1995). A similar pattern in the epithelia of mature guinea pigs. J Comp Neurol 397:69–88. Goldberg JM, Lysakowski A, Fernańdez C. 1992. Structure and function of mammalian vestibular epithelia might explain why only vestibular nerve fibers in the chinchilla and squirrel monkey. Ann NY bouton-innervated hair cells are observed in regenerating Acad Sci 656:92–107. epithelia (Tanyeri et al., 1995; Lopez et al., 1997). The Gray AA. 1907. The labyrinth of animals, vol 1. Including mammals, birds, suggested continuous development of the mammalian ves- reptiles and amphibians. London: Churchill. p 68–73 tibular hair cell is illustrated in Figure 6. Gundersen HJG. 1977. Notes on the estimation of numerical density of arbitrary profiles: the edge effect. J Microsc 111:219–223. Gundersen HJG. 1986. Stereology of arbitrary particles. A review of unbi- CONCLUSIONS ased number and size estimators and the presentation of some new ones. J Microsc 143:3–45. The present study confirms that the macular epithelia Igarashi M, Watanuki K, Miyata H, Alford B. 1975. Vestibular mapping in of the Daubenton’s bat exhibit ultrastructural features the squirrel monkey. Arch Otorhinolaryngol 211:153–161. that are basically mammalian. However, the ratio be- Iwata N. 1924. U¨ ber das Labyrinth der Fledermaus mit besonderer Be- tween the areas of the two macular epithelia is different ru¨ cksichtigung des statischen Apparates. Aichi J Exp Med 1:41–173. from that of other mammals so far investigated, a feature Jacobson MD, Weil M, Raff MC. 1997. Programmed cell death in animal that remains unexplained. The hair cells with immature development. Cell 88:347–354. morphology found in both maculae proved to be bouton- Jensen JC, Jørgensen JM. 2001. Dark hair cells in the inner ear of the innervated and indicate that the vestibular epithelia in rainbow trout. A study of the influence of different fixation methods. the mature bat have a low continuous generation of hair Acta Zool (Stockh) 82:79–88. Jørgensen JM. 1988. The number and distribution of calyseal hair cells in cells. In addition, the observation of several intermediate the inner ear utricular macula of some reptiles. Acta Zool (Stockh) types of hair cells suggests that the bouton- and chalice- 69:169–175. innervated hair cells are representatives of different de- Jørgensen JM. 1989. Number and distribution of hair cells in the utricular velopmental stages. The possible ongoing production of macula of some avian species. J Morphol 201:187–204. hair cells and the continuous development from bouton- to Jørgensen JM. 1991. Regeneration of lateral line and inner ear vestibular chalice-innervated hair cell are probably not unique to the cells. Regeneration of Vertebrate Sensory Receptor Cells/Ciba Symp Daubenton’s bat. Comparative studies of other vestibular 160:151–170. epithelia will reveal whether it is a common mammalian Jørgensen JM, Locket NA. 1995. The inner ear of the echidna Tachyglossus feature. aculeatus: the vestibular sensory organs. Proc R Soc Lond 260:183–189. Jørgensen JM, Mathiesen C. 1988. The avian inner ear: continuous production of hair cells in vestibular sensory organs, but not in auditory ACKNOWLEDGMENTS papilla. Naturwissenschaften 75:319–320. Kil J, Warchol ME, Corwin JT. 1997. Cell death, cell proliferation and We are grateful for the assistance of Sonja Kornerup in estimates of hair cell life spans in the vestibular organs of chicks. sectioning and staining the ultrathin sections. The assis- Hearing Res 114:117–126. tance of Birger Jensen in collecting the bats and of Hans Kirkegaard M, Jørgensen JM. 2000. Continuous hair cell turnover in the inner ear vestibular organs of a mammal, the Daubenton’s bat (Myotis Baagøe in X-raying and age-determining the bats is much daubentonii). Naturwissenschaften 87:83–86. appreciated. Thanks to Jens R. Nyengaard for providing Kuntz AL, Oesterle EC. 1998. Transforming growth factor ␣ with insulin the microscope and software for the area and density stimulates cell proliferation in vivo in adult rat vestibular sensory estimations. Finally, we are grateful for the valuable com- epithelium. J Comp Neurol 399:413–423. ments on the manuscript from Peter Teglbjerg Madsen, Lambert PR, Gu R, Corwin JT. 1997. Analysis of small hair bundles in the Stig Åvall Severinsen, Bjarke Klit Nielsen, and Birgitte utricles of mature guinea pigs. Am J Otol 18:637–643. Dahl. Lang H, Liu C. 1997. Apoptosis and hair cell degeneration in the vestibular sensory epithelia of guinea pig following a gentamicin insult. Hearing Res 111:177–184. LITERATURE CITED Li L, Forge A. 1997. Morphological evidence for supporting cell to hair cell conversion in the mammalian utricular macula. Int J Dev Neurosci Alison MR, Sarraf CE. 1992. Apoptosis: a gene-directed programme of cell 15:433–446. death. J R Coll Phys Lond 26:25–35. Li L, Nevill G, Forge A. 1995. Two modes of hair cell loss from the Baagøe HJ. 1977. Age determination in bats (Chiroptera). Videnskabelige vestibular sensory epithelia of the guinea pig inner ear. J Comp Neurol Meddelelser dansk naturhistorisk Forening 140:53–92. 355:405–417. Bagger-Sjo¨ba¨ck D, Gulley RL. 1979. Synaptic structures in the type II hair Li L, Forge A, Nevill G. 1997. Apoptotic death of hair cells. Assoc Res cell in the vestibular system of the guinea pig. Acta Otolaryngol Otolaryngol Abstr 20:445. (Stockh) 88:401–411. Lindeman HH. 1969. Studies on the morphology of the sensory regions of Baird RA, Steyger PS, Schuff NR. 1996. Mitotic and nonmitotic hair cell the vestibular apparatus. Adv Anat Embryol Cell Biol 42:1–113. regeneration in the bullfrog vestibular otolith organs. Ann NY Acad Sci Lindeman HH, Ades HW, West RW. 1973. Scanning electron microscopy of 781:59–70. the vestibular end organs. In: Proceedings of the 5th Symposium on the Beresford WA. 1990. Direct transdifferentiation: can cells change their Vestibular Organs in Space Exploration. Washington: NASA. p 145– phenotype without dividing? Cell Differ Dev 29:81–93. 156. Dechesne C, Mbiene JP, Sans A. 1986. Postnatal development of vestibular Lindenlaub T, Burda H, Nevo E. 1995. Convergent evolution of the vestib- receptor surfaces in the rat. Acta Otolaryngol (Stockh) 101:11–18. ular organ in the subterranean mole-rats, Cryptomys and Spalax,as Eguchi G, Kodoma R. 1993. Transdifferentiation. Curr Opin Cell Biol compared with the aboveground rat, Rattus. J Morphol 224:303–311. 5:1023–1028. Lopez I, Honrubia V, Lee SC, Shoeman G, Beykirch K. 1997. Quantifica- Engstro¨m H. 1961. The innervation of the vestibular sensory cells. Acta tion of the process of hair cell loss and recovery in the chinchilla crista Otolaryngol (Stockh) Suppl 163:30–40. ampullaris after gentamicin treatment. Int J Dev Neurosci 15:447– Favre D, Sans A. 1979. Embryonic and postnatal development of afferent 461. innervation in cat vestibular receptors. Acta Otolaryngol (Stockh) 87: Lysakowski A. 1996. Synaptic organization of the crista ampullaris in 97–107. vertebrates. Ann NY Acad Sci 781:164–182. 444 M. KIRKEGAARD AND J.M. JØRGENSEN

3Mbiene JP, Favre D, Sans A. 1984. The pattern of ciliary development in monkeys. The role of vestibular organs in the exploration of space. fetal mouse vestibular receptors. Anat Embryol 170:229–238. Washington, DC: NASA. p 7–22. Nakagawa T, Yamane H, Takayama M, Sunami K, Nakai Y. 1998. Apo- Takumida M, Harada Y. 1984. Development of the utricular macula in the ptosis of guinea pig cochlear hair cells following aminoglycoside treat- mouse. Arch Otorhinolaryngol 241:9–15. ment. Eur Arch Otorhinolaryngol 225:127–131. Tanyeri H, Lopez I, Honrubia V. 1995. Histological evidence for hair cell Nordemar H. 1983. Postnatal development of the vestibular sensory epi- regeneration after ototoxic cell destruction with local application of thelium in the mouse. Acta Otolaryngol (Stockh) 96:447–456. gentamicin in the chinchilla crista ampullaris. Hearing Res 89:194– Nyengaard JR. 1999. Stereological methods and their application in kidney 202. research. J Am Soc Nephrol 10:1100–1123. Tilney LG, Cotanche DA, Tilney MS. 1992a. Actin filaments, stereocilia Park JC, Hubel SB, Woods AD. 1987. Morphometric analysis and fine and hair cells of the bird cochlea. VI. How the number and arrange- structure of the vestibular epithelium of aged C57BL/6Nnia mice. ment of stereocilia are determined. Development 116:213–226. Hearing Res 28:87–96. Tilney LG, Tilney MS, DeRosier DJ. 1992b. Actin filaments, stereocilia and Ramprashad F, Landolt JP, Money KE, Laufer J. 1980. Neuromorphomet- hair cells: how cells count and measure. Annu Rev Cell Biol 8:257–274. ric features and dimensional analysis of the vestibular end organ in the Tsue TT, Oesterle EC, Rubel EW. 1994. Diffusible factors regulate hair cell little brown bat (Myotis lucifugus). J Comp Neurol 192:883–902. regeneration in the avian inner ear. Proc Natl Acad Sci USA 91:1584– Roberson DF, Weisleder P, Bohrer P, Rubel EW. 1992. Ongoing production 1588. of sensory cells in the vestibular epithelium of the chick. Hearing Res Warchol ME, Lambert PR, Goldstein BJ, Forge A, Corwin JT. 1993. Re- 57:166–174. generative proliferation in inner ear sensory epithelia from adult Rosenhall U. 1972. Vestibular macular mapping in man. Ann Otol Rhinol guinea pigs and humans. Science 259:1619–1621. Laryngol 81:339–351. Watanuki K, Meyer zum Gottesberge A. 1971. Morphologie der Sinnesep- Rosenhall U. 1973. Degenerative patterns in the aging human vestibular itelien des Vestibularorgans. Tohoku J Exp Med 104:55–63. neuro-epithelia. Acta Otolaryngol (Stockh) 76:208–220. Watanuki K, Stupp HF, Meyer zum Gottesberge A. 1971. Distribution Rubel EW, Dew LA, Roberson DW. 1995. Mammalian vestibular hair cell pattern of type I and type II sensory cells on the maculae sacculi and regeneration. Science 267:701–707. utriculi in the guinea pig. Pract Otorhinolaryngol 33:304–311. Schober W, Grimmberger E. 1987. A guide to bats of Britain and Europe. Weisleder P, Rubel EW. 1992. Hair cell regeneration in the avian vestib- London: Hamlyn Publishing Group. ular epithelium. Exp Neurol 115:2–6. Siegel S, Castellan NJ. 1988. Nonparametric statistics for the behavioral Weisleder P, Tsue TT, Rubel EW. 1995. Hair cell replacement in avian sciences, 2nd ed. New York: McGraw-Hill. pp 213–215. vestibular epithelium: supporting cell to type I hair cell. Hearing Res Sobkowicz HM, August BK, Slapnick SM. 1996. Post-traumatic survival 82:125–133. and recovery of the auditory sensory cells in culture. Acta Otolaryngol Wyllie AH. 1981. Cell death: a new classification separating apoptosis from (Stockh) 116:257–262. necrosis. In: Bowen JD, Lockshin RA, editors. Cell death in biology and Sobkowicz HM, August BK, Slapnick SM. 1997. Cellular interactions as a pathology. London: Chapman & Hall. p 9–34. response to injury in the organ of corti in culture. Int J Dev Neurosci Zheng JL, Keller G, Gao W-Q. 1999. Immunocytochemical and morpholog- 15:463–485. ical evidence for intracellular self-repair as an important contributor to Spoendlin H. 1965. Ultrastructural studies of the labyrinth in squirrel mammalian hair cell recovery. J Neurosci 19:2161–2170.