The Inner Ear Macular Sensory Epitheliaof the Daubenton’s Bat

METTE KIRKEGAARD AND JØRGEN MØRUP JØRGENSEN*

Department of Zoophysiology, University of Aarhus, DK-8000 Aarhus C, Denmark

ABSTRACTThe 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 ofthe sensory epithelium reveals two main types of hair cells: the chalice-innervated hair celland the bouton-innervated hair cell. The existence of an intermediate type, with a nerveending covering the lateral side of the hair cell, indicates that the chalice-innervated haircells 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 apoptotichair cells were observed. It is suggested that a continuous production of new hair cells takesplace 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 thathave developed active flight. It can be speculated thatmammals moving in three dimensions (i.e., flying or swim-ming) could exhibit special conformations in the vestibu-lar apparatus compared with their terrestrial counter-parts. Previous investigations have shown that the grossmorphology of the chiropteran vestibular apparatus istypical mammalian (Gray, 1907; Iwata, 1924; Ram-prashad et al., 1980). However, according to the mostrecent investigation, the areas of the sensory maculae inthe little brown bat (Myotis lucifugus) are very smallcompared with those of other mammals (Ramprashad etal., 1980). Additionally, the density of hair cells in themacula utriculi was found to be extremely high, whereasthe macula sacculi contained comparatively fewer haircells. The density of hair cells in the maculae is consistentwithin most mammalian species (Lindeman, 1969; Lin-denlaub et al., 1995), but the investigation by Ram-prashad et al. (1980) indicates that the sensory epitheliaof bats might differ from that of other mammals in thisrespect.

Since the finding that ongoing hair cell formation existsin normal avian vestibular sensory epithelia (Jørgensenand Mathiesen, 1988; Roberson et al., 1992), researchershave wondered whether the mammalian vestibular sen-sory epithelia may possess the same ability. Developingvestibular hair bundles have been observed in matureguinea pigs (Forge et al., 1993; Rubel et al., 1995; Lambertet al., 1997), but innervation was not confirmed. Whetherformation of new hair cells in vestibular sensory epithelia

is a general mammalian feature is not known. The originof the new hair cells is obscure because undamaged mam-malian vestibular epithelia show practically no cell divi-sions (Jørgensen, 1991; Rubel et al., 1995; Kuntz andOesterle, 1998). In this respect, bats are interesting ani-mals. They grow old compared with mammals of similarsize, and many of the Myotis species reach a maximum ageof 20 years and have average life spans of 3–5 years(Schober and Grimmberger, 1987). Furthermore, they aredependent on vestibular function due to their mode of life.Thus, if any mammal should exhibit a continuous produc-tion of hair cells in the vestibular organs, the bat is a likelycandidate.

In the present study, the maculae of the Daubenton’sbat (Myotis daubentonii, Kuhl 1819) were studied withemphasis on the utricular macula. The study had twomain objectives, the first of which was to investigate thevestibular epithelia of a microchiropteran species in rela-tion to size, structure, and number of hair cells. Becausedata on equal sized mammals are lacking, the vestibularsensory epithelia of the common shrew (Sorex araneus, L.)were also investigated. The second aim was to look for any

*Correspondence to: Jørgen Mørup Jørgensen, Department of Zoophysi-ology, Building 131, University of Aarhus, DK-8000 Aarhus C, Denmark.E-mail: joergen.moerup.joergensen@biology.au.dk

Received 25 February 2000; Revised 18 May 2001; Accepted 25 June2001

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

© 2001 WILEY-LISS, INC.

indications of turnover, i.e., developing and dying haircells, in the macular epithelia of adult bats.

MATERIALS AND METHODS

Ten specimens of Daubenton’s bat (M. daubentonii Kuhl1819) were collected at their winter quarters in Octoberand February. After decapitation, the bony labyrinth wasremoved, opened, and fixed in 3% glutaraldehyde in 0.15M phosphate buffer (pH 7.2) for 3 hours at room temper-ature. After a rinse in phosphate buffer, the material waspostfixed in 1% OsO4 in phosphate buffer for 1 hour,rinsed in phosphate buffer, and left in 70% ethanol over-night. The next day the vestibular sensory areas weredissected in 70% ethanol and dehydrated in a gradedseries of ethanol.

Maculae utriculi from three left ears and one right earwere mounted on glass slides in XAM, neutral medium.The right and left maculae sacculi from one specimen werelikewise mounted on glass slides. The total number of haircells was determined for each sensory epithelium by usinga BX-50 Olympus microscope. Images were captured byusing a 3-CCD camera. Counting was performed withCAST 2.0 software (Olympus Denmark A/S, Albertslund,Denmark) by using the 2D-fractionator (Gundersen,1986). The 2D unbiased counting frame (Gundersen, 1977)had an area of 542.5 mm2 and was moved across the entiresensory epithelium in steps of 45 mm in both the horizon-tal and the vertical directions. The total number of haircells is calculated as

N 5dx3dyAframe

3O Q2

where dx and dy are the distance between counting framesin horizontal and vertical directions (45 mm), Aframe is thearea of the counting frame (542.5 mm2), and Q2 isthe number of hair cells counted in each counting frame.The area was estimated by counting how many corners ofthe counting frame were found within the sensory epithe-lium at each sample point and calculated as

A 5dx3dy

p 3O P

where p is the maximal number of corners (4) and P is thenumber of corners counted in each sample point. Thecoefficient of error (CE) was calculated as

CE 51

ÎOQ2

and found to be between 0.04 and 0.05 for all estimates(Nyengaard, 1999). A mean number of 43 frames werecounted on maculae utriculi, and a mean number of 69frames were counted on maculae sacculi in Daubenton’sbat. The corresponding numbers for the common shreware 107 and 71, respectively. The average number of cellsper frame was 10 in all specimens. The sensory epitheliumwas discerned by the round apical surfaces of the haircells. Because the striola could not be discerned in thewhole mount preparations, no separate counts were madefor the striola. The hair cell density was calculated for

each sensory epithelium by using the estimated area andthe total hair cell number.

The sensory epithelia used for scanning electron micros-copy were fixed as described above. Next the material wascritical point dried, mounted on stubs, covered with plat-inum in a sputtercoater, and examined in the scanningelectron microscope (Maxim 2040, CamScan Electron Op-tics Ltd, Cambridge, U.K.) at 20 kV. By exposing thematerial to ultrasound prior to the critical point drying,the hair bundles were removed from the hair cell surfaces.The exact number of stereovilli was obtained by countingthe stubs at the apical cell surface. Counts were made on150 cell surfaces distributed over the pars externa, parsinterna, and striola. The striola was recognized by theorientation of the hair bundles, and countings were madewithin two hair cells on each side of the dividing line. Thetotal numbers of stereovilli per hair bundle as well as thenumber of rows from the kinocilium and the number ofstereovilli per row were counted. The averages for the parsexterna, pars interna, and striola were compared by usingthe Kruskal-Wallis test for k-independent samples. Posthoc comparisons between areas were made due to signif-icantly different counts (Siegel and Castellan, 1988).

The remaining material was embedded in Epon 812. Fortransmission electron microscopy the epon blocks were sec-tioned on an ultramicrotome (L. K. B. Ultrotome IV), pickedup on Formvar-coated copper grids, and stained with uranylacetate and lead citrate. One macula sacculi and one maculautriculi were cut tangential to the surface of the epithelium.One macula sacculi and two maculae utriculi were cut per-pendicular to the surface of the epithelium. The sectionswere examined in a transmission electron microscope (Zeiss10B, Zeiss Oberkochen, Germany). From two series cut per-pendicular to the surface, the thicknesses of the stereovilliand the cuticular plate were measured for the two hair celltypes. Sixty stereovilli were measured from eight randomlychosen chalice-innervated hair cells. On average, eight ste-reovilli were measured from each hair cell. From six ran-domly chosen bouton-innervated hair cells, 39 stereovilliwere measured. On average, six stereovilli were measuredfrom each hair cell. The thickness was measured as thediameter of the stereovillus at a height two-thirds of the totalheight. The measurements from chalice-innervated andbouton-innervated hair cells were compared as individualobservations by using a t-test. The thicknesses of cuticularplates from six randomly chosen chalice-innervated haircells and seven randomly chosen bouton-innervated haircells were measured and compared by using a t-test.

For light microscopy, the blocks were cut into 2-mmsections, mounted on glass slides, and stained with tolu-idine blue. From tangential sections, the ratio betweenchalice-innervated and bouton-innervated hair cells wascounted in two different maculae. The striola was definedas two hair cells on each side of the line dividing hair cellswith opposite orientation. Hair cells were counted withinan area of 50 3 50 mm. Four samples were taken from thestriola, and eight samples were taken from the extrastriolar areas.

To separate young animals from old ones, the degree offusion of epiphyses in the distal part of the metacarpal ofthe third finger was determined from X-ray radiographs.The young, born in July, can by this method be discernedfrom adults until the following spring (Baagøe, 1977). Allanimals except two exhibited complete fusion of epiphysesand were thus classified as adults. The remaining two

434 M. KIRKEGAARD AND J.M. JØRGENSEN

animals showed traces of epiphyseal closure, but becausethey 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 werefixed in 3% glutaraldehyde for between 3 and 24 hours butwere otherwise treated as previously described. The utric-ular maculae from five different animals were mounted onglass slides in XAM, neutral media. Likewise, the saccularmaculae from three different animals were mounted onglass slides.

All animals were handled in accordance with NIH rules.Permission to collect and sacrifice bats was given by theNational Forest and Nature Agency, Denmark.

RESULTS

Gross structure

The bony labyrinth of the Daubenton’s bat is isolatedfrom the temporal bone by connective tissue. The mem-branous labyrinth contains five sensory organs in the ves-tibular part and the organ of Corti in the cochlea. Each ofthe three semicircular ducts has an ampulla containing acrista. A transverse bar covered by nonsensory epithelia,the eminentiae cruciatae, divides the anterior and poste-rior cristae into two approximately equal parts. The threeampullae open into the utriculus, which contains the mac-ula utriculi. Below the utricular recessus is the sacculus,containing the macula sacculi. The macula sacculi is po-sitioned in a sagittal plane perpendicular to the maculautriculi. The maculae are overlaid by an otoconial mem-brane, which follows the outline of the sensory epithelium.

Quantitative data for the maculae of the Daubenton’sbat are given in Table 1.The area of the macula utriculiwas found to vary between 0.049 mm2 and 0.090 mm2,with a mean of 0.069 6 0.01 mm2 (mean 6 SE; n 5 4). Themean total number of sensory cells in the macula was1307 6 136 (n 5 4). The outline of a representative utric-ular macula is given in Figure 1. The mean area of themaculae sacculi from one animal was found to be 0.145 60.001 mm2 (n 5 2), and the mean total number of cells was2639 6 80 (n 5 2). For comparison, the corresponding datafor the common shrew are given in Table 2.

Structure of the sensory epithelium

The macular sensory epithelium contains supportingcells, nerve fibers, and two types of sensory cells. Thesupporting cells span the height of the entire epithelium,and their irregular nuclei are situated close to the basal

lamina. At the luminal surface, they have numerous mi-crovilli and a central ciliary rod (Fig. 2b). The supportingcells possess a reticular membrane seen as a dark bandbelow the apical surface (Fig. 2a,b). The chalice-innervated hair cell (type I) is recognized by an afferentnerve ending forming a chalice around the basolateralpart of the cell (Figs. 2a, 4c). A constriction below thecuticular plate forms a neck and gives the cell a charac-teristic flask-shaped appearance. In the apical region, nu-merous mitochondria are seen. The bouton-innervatedhair cell (type II) is ovoid to columnar in shape. It isinnervated by afferent and efferent nerve endings formingboutons at the basal parts of the cell (Figs. 2a, 4c).

In the cytoplasm, numerous mitochondria and vesiclesare seen. Both cell types exhibit synaptic structures op-posed to the nerve endings. These synaptic bodies occurindividually or in pairs and may be spherical or elongatedbars. The nuclei of the chalice-innervated hair cells arelocated in the lower part of the epithelium, above thesupporting cell nuclei, whereas the nuclei of the bouton-innervated hair cells are generally located more luminally(Fig. 2a). Both types of hair cells have an apical hairbundle consisting of an eccentrically located kinocilium

TABLE 2. Sorex: Data on Maculae Utriculi and Sacculi

Specimen1 Area (mm2)Total hair cell

populationHair cell density

(cells/mm2)

Macula utriculi1R 0.2202R 0.101 1810 17,8803R 0.198 3233 16,2893L 0.210 3986 18,975Mean 6 SE 0.182 6 0.031 3010 6 781 17,715 6 955

Macula Sacculi3R 0.1503L 0.129 2165 16,7716L 0.111 1885 16,925Mean 6 SE 0.130 6 0.014 2025 6 198 16,848 6 109

1R, right ear; L, left ear.

TABLE 1. Myotis: Data on the Maculae Utriculi and Sacculi

Specimen1Weight

(g)Area

(mm2)

Totalhair cell

population

Hair celldensity

(cells/mm2)

macula utriculi2L 12.31 0.071 1355 18,9824L 10.21 0.049 982 19,7475R 9.73 0.090 1545 17,1496L 10.50 0.066 1347 20,475Mean 6 SE 10.69 6 0.65 0.069 6 0.010 1307 6 136 19,098 6 829

macula sacculi5R 9.73 0.146 2696 18,4885L 0.144 2583 17,902Mean 6 SE 0.145 6 0.001 2639 6 80 18,195 6 414

1R, right ear; L, left ear.

Fig. 1. Outline of the left macula utriculi of the Daubenton’s bat.The orientation of the hair bundles is indicated with arrows. Thearrow point indicates the position of the kinocilium relative to thestereovilli. A, anterior direction; m, medial direction; pi, pars interna;pe, pars externa; s, striola. Scale bar 5 100 mm.

435THE MACULAR SENSORY EPITHELIA OF THE BAT

Fig. 2. Transmission electron micrographs of the epithelium ofmacula utriculi showing the two types of hair cells. a: A chalice-innervated hair cell and a bouton-innervated hair cell. The bouton-innervated hair cell is contacted by a large nerve ending (n) coveringthe lateral side of the cell and a conventional bouton (*). The chalice-innervated hair cell is recognized by a narrow neck and a nervechalice (nc) surrounding the basolateral part of the cell. The reticular

membrane (rm) of the supporting cells is seen as a dark band belowthe epithelial surface. b: The hair bundles of a bouton-innervated (B)and a chalice-innervated (C) hair cell. The latter has thick stereovilliwith flat tips and a dense cuticular plate. One supporting cell dem-onstrates the ciliary rod (arrow). c: The hair bundle of a chalice-innervated hair cell. The stereovilli have flat tips and anchors withfine rootlets in the cuticular plate (arrow). Scale bars 5 2 mm.

and a number of stereovilli arranged in rows of decreasingheight from the kinocilium. Each stereovillus anchorswith a fine rootlet in the cuticular plate (Fig. 2c). However,there are differences between the hair bundles of the twocell types. The stereovilli of the chalice-innervated haircells are thicker than the stereovilli of the bouton-innervated hair cells, and they have flat tips. The stere-ovilli of the chalice-innervated hair cells were estimated tohave a diameter of 0.168 6 0.004 mm (n 5 60), and thestereovilli of the bouton-innervated hair cells were esti-mated to have a diameter of 0.128 6 0.003 mm (n 5 39),but this difference was not found to be significant. Fur-thermore, the cuticular plate of the chalice-innervatedhair cells appears thicker and denser than the cuticularplate of the bouton-innervated hair cell, but a statisticallysignificant difference in the thickness of the cuticularplate could not be confirmed (0.783 6 0.085 mm vs. 0.689 60.143 mm [n 5 6, n 5 7]; Fig. 2b).

The bouton-innervated hair cells vary from a sphericalto a cylindrical, almost flask-shaped type of cell, resem-bling the chalice-innervated cells. In addition to the affer-ent boutons innervating the hair cells basally, there arelaterally situated synapses on both bouton- and chalice-innervated hair cells. Some of the bouton-innervated haircells are innervated by large flat boutons that cover asubstantial part of the lateral side of the cell (Fig. 2a).

The striola, recognized by the orientation of the hairbundles, runs parallel to the anterolateral margins anddivides the sensory epithelium into a medial pars internaand a lateral pars externa (Fig. 1). The striola is domi-nated by chalice-innervated hair cells, the chalice type/bouton type ratio being 3.9:1. In the extrastriolar areas,the same ratio is 1.3:1. In the striola, the chalice-innervated hair cells occur in chalices enclosing one, two,or three cells, whereas the extrastriolar region mainlycontains chalices with a single hair cell.

Supposed immature hair bundles were observed in allutricular and saccular maculae investigated. In tangentialsections they are recognized by a hair bundle consisting ofa central kinocilium surrounded by thin stereovilli (Fig.3). Eleven immature hair bundles were found in a singleutricular macula, but due to incomplete series, the totalnumber per macula could not be obtained. In the remain-ing maculae, between two and five immature hair bundleswere observed. Most of the immature hair bundles werefound close to the striola and occurred in clusters (Fig. 3).In sections cut perpendicular to the surface of the sensoryepithelium, the position of the kinocilium was difficult tolocate. However, some of the bouton-innervated hair cellsdiffered from the type described above. The cell bodyspans a large part of the epithelium, but no contact withthe basal lamina was observed. The cuticular plate isindistinct or absent, and mitochondria are visible in thesupranuclear region. Some of these hair cells have anirregular/oval nucleus located basally whereas othershave the nucleus situated in the central region of the cellbody. They are innervated basally with bouton nerve end-ings. These hair cell bodies are considered to belong to theimmature hair bundles (Fig. 4c).

In ultrathin sections from all four maculae investigated,apoptotic cells were observed in the sensory epithelium.They have a dark staining (pyknotic) nucleus, and the cellplasma is vesiculated and condensed. Apoptosis was ex-clusively seen in single chalice-innervated hair cells or inhair cells in chalices shared with normal cells (Fig. 4a,b).

However, there was no quantitative investigation of theoccurrence of apoptotic cells due to incomplete series.

Size and shape of hair bundles

The number of stereovilli per hair bundle varies withinthe sensory epithelium. In the striola, the mean number ofstereovilli per hair bundle is significantly lower comparedwith the extrastriolar areas (35.4 6 0.9 (n 5 52) vs. 53.7 61.4 (n 5 89); p , 0.01). This difference is due to fewer rowsof stereovilli in the striolar hair bundles (3.8 6 0.13 vs.6.3 6 0.16; p , 0.01), whereas the number of stereovilliper row is the same in the striola and the extrastriolarareas (10.0 6 0.19). The limited number of rows gives thestriolar hair bundles a distinctive flattened appearance(compare Fig. 3a and b). From tangential sections throughthe epithelium, it is evident that the flat hair bundles inthe striola belong to chalice-innervated hair cells, whereasthe chalice-innervated hair cells in the extrastriolar areashave normal looking hair bundles.

DISCUSSION

Size of the sensory epithelia

The area of the macula utriculi of the Daubenton’s batwas estimated to be 0.069 mm2 and the area of the maculasacculi to be 0.145 mm2. The estimate for the saccularepithelium should be viewed with reservations, because itis based on the right and left sensory epithelia from oneanimal. In the study by Ramprashad et al. (1980), thecorresponding areas in the little brown bat (M. lucifugus)were estimated to be 0.016 mm2 and 0.098 mm2, respec-tively. These results agree with the present study in twoways: the macular sensory epithelia are very small, andthe saccular macula is considerably larger than the utric-ular macula. However, estimates of the macular areas andthe total hair cell population differ in the two bat species,which are of equal size—a fact that may be due to thedifferent methods used. The present study concludes thatthe vestibular apparatus of the Daubenton’s bat is typi-cally mammalian and that the extreme relations reportedin the sensory epithelia of the little brown bat do notpertain to the Daubenton’s bat.

When comparing the area of the maculae in theDaubenton’s bat with the common shrew, an animal ofequal size, the maculae utriculi of the common shrewproved to be more than twice the area of the correspondingsensory epithelium in the Daubenton’s bat, whereas thesaccular sensory epithelia were of equal size (Tables 1, 2).To investigate whether this difference is due to a deviationin one or the other of the two species, they were comparedwith available data for other mammals. Data on guineapig (Lindeman, 1969), two mole rat species, and rat (Lin-denlaub et al., 1995) are shown together with results fromthe present study (bat and shrew) in Table 3. All the dataincluded rely on whole mount preparations. The largerelative size of the sensory epithelia in bat and shrew mayreflect their small size. One might expect the area of thesensory epithelia to be roughly correlated with size (i.e.,weight). However, if minimum numbers of sensory recep-tors are required for adequate sensitivity/resolution, theremay be a lower limit to the total number of hair cells,which implies a minimum size of the epithelia. This wouldresult in small animals exhibiting relatively larger sen-sory epithelia. Due to the limited amount of available data

437THE MACULAR SENSORY EPITHELIA OF THE BAT

Fig. 3. Transmission electron micrographs of a tangential sectionof hair bundles in the macula utriculi. a: The extrastriolar area, parsexterna. Most hair bundles are oriented toward the striola, i.e., thekinocilium is located peripherally. Three immature hair bundles (ar-rows) have thin and unorganized stereovilli compared with the ma-ture hair bundles and a central kinocilium. b: Tangential section

through the striola. The hair bundles have fewer rows of stereovilli(compare with hair bundles in a). Two of the hair bundles have acentral kinocilium and are thus classified as immature (arrows). Ar-rows in lower right corner indicate orientation of both sections: A,anterior direction on the macula; M, medial direction on the macula.Scale bars 5 2 mm.

Fig. 4. a: Nerve chalice with an apoptotic cell co-occurring with a normal cell. The nucleus is pycnoticand the cytoplasm is vesiculated. b: Chalice-innervated apoptotic cell. c: Two chalice-innervated haircells and an immature hair cell. The latter has an oval nucleus and is contacted by two boutons (arrows).Scale bars 5 2 mm.

on other mammals, it is difficult to deduce any clear rela-tions between the area of the vestibular sensory epitheliaand animal size.

In the Daubenton’s bat and in the little brown bat (Ram-prashad et al., 1980), the saccular macula is considerablylarger than the utricular macula. This feature is illus-trated in Figure 5. In the graph, the area of the maculautriculi is plotted against the area of the macula sacculi.This reveals that in all the other mammalian species, the

ratio between the macular areas lies either close to orabove the line y 5 x, i.e., the macular sensory epithelia areof equal size or the utricular macula is larger than thesaccular macula. Because bats are the only flying mam-mals, a possible explanation could be that the large sac-cular macula reflects an adaptation to flight. It could besupposed that bats are subjected to vertical accelerationmore frequently than ground-living mammals and hencehave a well-developed vertical macula. If the deviation inthe chiropteran vestibular epithelia is a mammalian ad-aptation to the aerial mode of life, it would be obvious tocompare bats with marine mammals or primates with anarboreal lifestyle. Like flying animals, animals swimmingin water are expected to move equally in three dimen-sions, as opposed to terrestrial animals, which, in spite ofjumping/climbing, are mainly subjected to horizontal ac-celerations. Likewise, mammals living in trees are fre-quently subjected to vertical accelerations. However, inthe squirrel monkey, the utricular and saccular maculaeare of equal size (Igarashi et al., 1975), and there are noinvestigations of the exact dimensions of the sensory epi-thelia in marine mammals. It must be concluded that sofar there is no clear explanation of why the saccular mac-ula is larger than the utricular macula in bats.

Structure of the sensory epithelium

The macular sensory epithelia of the Daubenton’s batexhibit general mammalian features: bouton- and chalice-innervated hair cells surrounded by supporting cells and astriola in the utricular macula that is dominated bychalice-innervated hair cells. A striolar zone, dominatedby chalice-innervated cells, is known from other mam-mals, including guinea pig (Lindeman, 1969; Watanukiand Meyer zum Gottesberge, 1971; Watanuki et al., 1971),echidna (Jørgensen and Locket, 1995), and human (Rosen-hall, 1972). The ratio between chalice-innervated andbouton-innervated hair cells found in the striola of guineapig is approximately 2:1, whereas the extrastriolar areahas a ratio of 1:1 (Lindeman, 1969; Watanuki and Meyerzum Gottesberge, 1971; Watanuki et al., 1971). In thepresent study the striola is found to be almost exclusivelypopulated by chalice-innervated hair cells (ratio of chalice-innervated/bouton-innervated cells 5 3.9:1). Data fromother mammalian maculae are lacking, but the ratio ofchalice-innervated/bouton-innervated hair cells is knownto vary in the mammalian cristae (3:1 in squirrel monkey;1:1 in chinchilla; Goldberg et al., 1992). The number ofcells per chalice is lower in mammals than in the otheramniotes. In the guinea pig, two to three hair cells can befound in the same chalice in the striola (Watanuki andMeyer zum Gottesberge, 1971; Watanuki et al., 1971),which corresponds well to the findings of the present studyin which some chalices were found to contain two or threecells. In birds, the chalices contain up to 10 cells perchalice (Jørgensen, 1989), whereas the reptilian chalicesenclose 1–5 cells, 1–3 being the most common (Jørgensen,1988).

Lindeman (1969) and Lindeman et al. (1973) have pre-viously noted the flat hair bundles in the striola of theutricular macula in guinea pig. They assumed that thehair bundles belong to chalice-innervated hair cells, due toa large free surface area and club-shaped stereovilli. Inthe present study, the hair cells with flattened hair bun-dles are followed through the epithelium in serial sections.These tangential sections verify that the flat hair bundles

Fig. 5. The area of macula utriculi plotted against the area ofmacula sacculi (mm2). The macular ratios are close to the line y 5 xexcept for the two bat species, which have large saccular maculasrelative to the utricular maculas. Solid square, little brown bat (Myo-tis lucifugus; data from Ramprashad et al., 1980); open square,Daubenton’s bat (Myotis daubentonii; data from the present study);solid diamond, common shrew (Sorex araneus; data from the presentstudy); open diamond, rat (Rattus norvegicus; data from Lindenlaubet al., 1995); solid triangle, Zambian common mole rat (Cryptomys sp.;data from Lindenlaub et al., 1995); open triangle, blind mole rat(Spalax ehrenbergi; data from Lindenlaub et al., 1995); solid circle,guinea pig (Cavia sp.; data from Lindeman, 1969).

TABLE 3. Data on Maculae Utriculi and Sacculi From DifferentMammalian Species

SpeciesWeight

(g)Area

(mm2)Relative

area1

Total haircell

population

Hair celldensity

(cells/mm2)

Macula utriculiMyotis 11 0.069 0.629 1307 18,885Sorex 12 0.182 1.520 3010 16,503Cryptomys 80 0.470 0.588 8100 17,234Spalax 120 0.509 0.424 8470 16,640Rattus 220 0.360 0.164 6020 16,722Cavia 300 0.541 0.180 9260 17,116

Macula sacculiMyotis 11 0.145 1.319 2639 18,197Sorex 12 0.130 1.083 2025 15,577Cryptomys 80 0.429 0.536 7040 16,410Spalax 120 0.483 0.403 7400 15,321Rattus 220 0.381 0.173 5960 15,643Cavia 300 0.495 0.165 7560 15,273

1Relative area ([area/weight] 3 100). Data on Myotis and Sorex are from the presentstudy. Data on Cryptomys, Spalax, and Rattus are from Lindenlaub et al. (1995). Dataon Cavia are from Lindeman (1969).

440 M. KIRKEGAARD AND J.M. JØRGENSEN

belong to chalice-innervated hair cells. Furthermore, thedifference in shape between hair bundles in the striolaand the extrastriola is quantified. It has been shown thatthe hair bundles in the striola have significantly fewerstereovilli and that this is due to significantly fewer rowsof stereovilli. This arrangement of the stereovilli gives thestriolar hair bundles a resemblance to the inner hair cellsin the organ of Corti. In the present study, the totalnumber of stereovilli per hair bundle was found to be 35 inthe striola and 54 in the extrastriolar areas. This is lowcompared with previous investigations, which report be-tween 50 and 100 stereovilli in guinea pig vestibular hairbundles (Lindeman, 1969) and between 60 and 100 stere-ovilli in the squirrel monkey (Spoendlin, 1965). The func-tional significance of these differences is unknown.

In the present study, the chalice-innervated hair cell isfound to possess thicker stereovilli and a more voluminouscuticular plate than the bouton-innervated hair cell, evenif these differences are not statistically significant. Simi-lar observations have been noticed in other amniotes (Ly-sakowski, 1996). This can be explained by the fact that thechalice-innervated hair cells are ontogenetically olderthan the bouton-innervated hair cells (see below).

Apoptotic cells in normal epithelia

Apoptotic cells are observed in the macular epithelia ofthe Daubenton’s bat. Apoptosis is a type of programmedcell death that occurs during tissue development and aspart of the turnover in normal tissue (Wyllie, 1981; Alisonand Sarraf, 1992; Jacobson et al., 1997). A low degree ofapoptosis is observed in normal mammalian vestibularsensory epithelia (Li et al., 1995; Kuntz and Oesterle,1998; Zheng et al., 1999). In the avian vestibular sensoryepithelia, the continuous production of hair cells (Jør-gensen and Mathiesen, 1988; Roberson et al., 1992;Weisleder and Rubel, 1992; Tsue et al., 1994; Weisleder etal., 1995) is probably regulated by apoptotic cell death, tomaintain a constant number of hair cells (Jørgensen,1991; Roberson et al., 1992; Kil et al., 1997). Additionally,apoptosis is recognized as a mode of hair cell degenerationfollowing aminoglycoside insult (Li et al., 1995, 1997;Lang and Liu, 1997; Nakagawa et al., 1998; Forge et al.,1998; Zheng et al., 1999). The apoptotic cells observed inthe present study are sparse and found scattered amonghealthy cells. This indicates that they are not the result ofchemical or mechanical damage during preparation, inaccordance with a recent examination of the fish inner ear(Jensen and Jørgensen, 2001). Unfortunately, it was notpossible to quantify the apoptotic cells with the presentmaterial.

Only chalice-innervated hair cells are recognized as ap-optotic. This may be a coincidence, because observations ofapoptotic hair cells are very sparse. However, this obser-vation supports the hypothesis of a continuous develop-ment from the bouton-innervated hair cell to the chalice-innervated hair cell (see below). Because the chalice-innervated hair cells are ontogenetically the oldest, theymay be expected to be more frequently subjected to apo-ptosis. The total number of hair cells declines with age inthe vestibular epithelia in humans (Rosenhall, 1973) andmice (Park et al., 1987). It is not known whether this is thecase in the vestibular epithelia of bats. However, it islikely that a long-lived animal such as the bat, dependingon vestibular function, should exhibit some regenerative

processes to compensate for the hair cells lost by apopto-sis.

Immature hair cells in mature animals

The bouton-innervated hair cells with a central kinoci-lium, observed in the present study, are interpreted asdeveloping hair cells. This is based on several descriptionsof the ontogenetic development of the vestibular sensoryepithelia in mouse and chick (Mbiene et al., 1984; Taku-mida and Harada, 1984; Dechesne et al., 1986; Tilney etal., 1992a,b) and descriptions from the vestibular sensoryepithelia in guinea pigs, regenerating after ototoxic dam-age (Forge et al., 1993, 1998; Rubel et al., 1995; Li andForge, 1997). These studies show that in the early stage ofhair bundle formation, numerous stereovilli of equalheight form around a central kinocilium on the apicalsurface of the hair cell. Later the kinocilium is found closeto the periphery, and the stereovilli attain decreasingheight with increased distance from the kinocilium. Next,the stereovilli widen by adding more actin filaments. Fi-nally, a cuticular plate develops, and the stereovilli elon-gate to the final height. This course of events correspondswell to the types of hair bundles observed in the presentstudy.

From a previous study (Kirkegaard and Jørgensen,2000), it is evident that when followed through the epi-thelium in a series of sections, the immature hair bundlesprove to belong to bouton-innervated hair cells. Theseimmature, bouton-innervated hair cells are believed tocorrespond to the subtype of bouton-innervated hair cellsobserved in sections cut perpendicular to the surface of thesensory epithelia. These hair cells have no visible cuticu-lar plate and are thus regarded as an early stage in thedevelopment.

Spoendlin (1965) observed hair bundles with a centralkinocilium in the vestibular epithelia of the squirrel mon-key but ascribed it to the irregular orientation in somehair bundles. Recently, similar hair bundles have beenobserved in mature guinea pigs (Forge et al., 1993; Rubelet al., 1995; Lambert et al., 1997; Forge et al., 1998). Thepresent study confirms the existence of hair cells with animmature morphology in a mature mammal and furtherthat the immature hair cells are innervated with afferentsynapses, which indicates normal function. We do notknow whether these hair cells differentiate any further.Alternatively, they could represent a subgroup of haircells not previously described.

Origin of immature hair cells

If the hair cells with immature morphology are devel-oping hair cells, their origin is obscure. The avian vestib-ular sensory epithelia are capable of continuous produc-tion of hair cells (Jørgensen and Mathiesen, 1988;Roberson et al., 1992; Tsue et al., 1994; Weisleder et al.,1995), but previous experiments with [3H]thymidine la-beling in mammalian vestibular epithelia show no prolif-eration (Jørgensen, 1991; Rubel et al., 1995; Kuntz andOesterle, 1998). Hence, it is doubtful whether the imma-ture hair cells observed in the present study are the re-sults of immediately preceding mitoses.

One possibility is that partially injured hair cells are re-growing a new hair bundle. It has been demonstrated thatafter sublethal damage (mechanical or ototoxic) in which thehair cell loses its hair bundle, it will relocate the kinociliumto the center of the cell surface and regrow a new hair bundle

441THE MACULAR SENSORY EPITHELIA OF THE BAT

(Sobkowicz et al., 1996, 1997; Zheng et al., 1999). This willresult in hair bundles with a morphology similar to the oneobserved in the present study. However, the elongated cellbody of the observed immature hair cells and the basallylocated nucleus imply that the deviation from mature haircells is not limited to the hair bundle.

An alternative explanation is that the developing haircells originate from supporting cells through transdiffer-entiation, which is the phenotypic conversion of a cell at alate developmental stage (Beresford, 1990; Eguchi andKodoma, 1993). Transdifferentiation has previously beensuggested in regenerating inner ear epithelia of the bull-frog (Baird et al., 1996) and the chick (Weisleder et al.,1995). In the regenerating mammalian epithelia, transdif-ferentiation has been considered, because the number ofnew hair cells observed in SEM disagrees with the num-ber of dividing cells observed in the epithelium after oto-toxic damage (Warchol et al., 1993; Rubel et al., 1995; Liand Forge, 1997). Studies by Li and Forge (1997) andForge et al. (1998) report morphological indications oftransdifferentiation in the regenerating vestibular epithe-lia of the guinea pig. Cells with supporting cell featuressuch as a reticular membrane and contact with the basallamina exhibited immature hair bundles.

In the present study, several morphological traits indi-cate transdifferentiation. Like the supporting cells, theimmature hair cells span a large part of the epithelium.The nuclei are irregular, and some cells have the nucleussituated basally, close to the supporting cell nuclei. It issuggested that because transdifferentiation of supportingcells seems to be part of the regeneration in damaged

mammalian sensory epithelia, it might well be a way ofrenewing hair cells in a continuous turnover. Of course,the production of new hair cells by transdifferentiation ofsupporting cells will eventually deplete the supporting cellpopulation. The population of supporting cells could berenewed by short periods of mitotic activity. This wouldexplain the difficulties in labeling mitoses in undamagedmammalian sensory epithelia.

Hair cell types

In the present study, intermediate types between thechalice- and bouton-innervated hair cell were observed.The cells innervated by boutons as well as by a large nerveending covering half of the cell (Fig. 2a) resemble descrip-tions from the ontogenetic development in the vestibularsensory epithelia of cat and mouse (Favre and Sans, 1979;Nordemar, 1983). From these studies, it is evident thatthe developing chalice-innervated hair cell passes througha stage where it resembles the bouton-innervated haircell. Nerve endings covering the lateral part of a bouton-innervated hair cell have previously been observed in ma-ture animals (Engstrom, 1961; Bagger-Sjoback and Gul-ley, 1979) and have been suggested to be a developmentalstage between the two types of hair cells (Engstrom,1961). Finally, the chalice-innervated hair cells havethicker stereovilli and a denser cuticular plate, whichindicate a later developmental stage (cf. the above descrip-tion of the ontogenetic development of the hair bundle).Together, these data imply that at least some of thebouton-innervated hair cells are merely an early develop-mental stage of the chalice-innervated hair cells. A con-

Fig. 6. Schematic outline of the suggested development of themammalian vestibular hair cell. A normal supporting cell with irreg-ular nucleus close to the basal lamina (1). The supporting cell losescontact with the basal lamina and becomes innervated by nerveendings. At the apical surface, stereovilli start to form around acentral kinocilium and the supporting cell is now considered an im-mature hair cell (2). When the hair bundle is formed and synapses

established, the hair cell is recognized as a bouton-innervated haircell (3). Some of the bouton-innervated hair cells are contacted bynerve endings, which enlarge and form a chalice (4). A further in-crease in the number of actin filaments results in thicker stereovilliand a denser cuticular plate. The final stage is a chalice-innervatedhair cell (5A). If the hair cell is situated in the striola, the hair bundleis reduced (5B). L, luminal surface; B, basal lamina.

442 M. KIRKEGAARD AND J.M. JØRGENSEN

tinuous development from a bouton-innervated hair cell toa chalice-innervated hair cell has previously been de-scribed in the regenerating vestibular epithelia of thechick (Weisleder et al., 1995). A similar pattern in themammalian vestibular epithelia might explain why onlybouton-innervated hair cells are observed in regeneratingepithelia (Tanyeri et al., 1995; Lopez et al., 1997). Thesuggested continuous development of the mammalian ves-tibular hair cell is illustrated in Figure 6.

CONCLUSIONS

The present study confirms that the macular epitheliaof the Daubenton’s bat exhibit ultrastructural featuresthat are basically mammalian. However, the ratio be-tween the areas of the two macular epithelia is differentfrom that of other mammals so far investigated, a featurethat remains unexplained. The hair cells with immaturemorphology found in both maculae proved to be bouton-innervated and indicate that the vestibular epithelia inthe mature bat have a low continuous generation of haircells. In addition, the observation of several intermediatetypes of hair cells suggests that the bouton- and chalice-innervated hair cells are representatives of different de-velopmental stages. The possible ongoing production ofhair cells and the continuous development from bouton- tochalice-innervated hair cell are probably not unique to theDaubenton’s bat. Comparative studies of other vestibularepithelia will reveal whether it is a common mammalianfeature.

ACKNOWLEDGMENTS

We are grateful for the assistance of Sonja Kornerup insectioning and staining the ultrathin sections. The assis-tance of Birger Jensen in collecting the bats and of HansBaagøe in X-raying and age-determining the bats is muchappreciated. Thanks to Jens R. Nyengaard for providingthe microscope and software for the area and densityestimations. Finally, we are grateful for the valuable com-ments on the manuscript from Peter Teglbjerg Madsen,Stig Åvall Severinsen, Bjarke Klit Nielsen, and BirgitteDahl.

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