Hearing Research, 11 (1983) 23-32

Elsevier

23

Ultrastructure of the lining of the Scala tympani of the bat, Pteronotus parnellii

D.B. Jenkins ‘, M.M. Henson ’ and O.W. Henson, Jr. ’

Departments of 'Anatomy and ‘Surgery, Uniuersrty of North Carolina, Chapel Hill, NC 27514, iJ.S.A

(Received 26 April 1982; accepted 24 February 1983)

The cells lining the Scala tympam of the cochlea of Pteronotur p. parnellii were studied in whole mount

preparations and with light and scanning and transmission electron microscopy. On the basis of structure

and location three different cell types were recognized: (1) those lying on the undersurface of the basilar

membrane; (2) those covering the internal surface of most of the otic capsule; and (3) those associated

with a thick layer of osmiophilic substance and restricted to a specific region in the basal turn. The cells

associated with the osmiophilic substance were strikingly different from the other cells; they were

relatively rich in organelles and had a Golgi complex which appeared to produce granules which coalesced

both intracellularly and extracellularly to form the osmiophilic layer. The function and composition of the

osmiophilic substance is unknown but it seems to be unique to Pteronotur parneffii and related subspecies

known to have greatly enlarged perilymphatic scalae and unusual hearing capacities associated with

Doppler shift compensation sonar.

Key words: cochlea; Scala tympani; bat.

Introduction

The cochleae of Pteronotus parnellii are known to have many morphological and physiological specializations associated with their remarkable sense of hearing

[ 10,l 1,14,16,20,2 l-23,28,29]. One striking characteristic is the size of the cochlea which, relative to the size of the animal, is much larger than that of any other species of bat and perhaps any other mammal. The size is created primarily by an enormous enlargement of the perilymphatic scalae. The Scala tympani is associated with a round window which is nearly as large as the tympanic membrane. A large cochlear aqueduct is also present and although it has an opening which faces into the cranial

cavity, it is closed by a membrane derived from the dura mater. Our studies [ 12,13,15], as well as those of Pye [24, 251 have shown that a portion

of the lining of the Scala tympani is associated with an unusual substance which is present as an osmiophilic layer. This layer, which may be as much as 50 pm thick, is restricted to a limited portion of the basal turn and extends through the cochlear aqueduct. According to Pye [25] the layer is invariably present in adult specimens but not in fetal or very young animals. On the basis of a hemoglobin-specific stain (Buffalo black) and the presence of clumps of material which resemble degenerated

0378-5955/83/$03.00 0 1983 Elsevier Science Publishers B.V

24

red blood cells, Pye has suggested that the substance composing the layer represents lysed erythrocytes and massed hemoglobin. However, our ultrastructural studies in many different regions of the basal turn, show that this substance is much more than a simple accumulation of the lysed cells or hemoglobin.

The purpose of this study was to examine the structure of the cells lining the Scala tympani (tympanic covering layer) in Pteronorus and to compare the ultrastructural features with those known for other mammals.

Materials and Methods

Mustache bats (Pteronotmparneliiiparnellii) from Jamaica, W.I. were used in this study. The animals were decapitated, the heads cut in the mid-sagittal plane and the cochleae rapidly removed. The cochleae were immersed in fixative consisting of 4% glutaraldehyde and 4% sucrose in 0.2 M s-co&dine buffer, pH 7.3. The apex of each cochlea was opened and the round window membrane was punctured to allow fixative to flow freely throughout the cochlea. The tissue remained in fixative 8-24 h; decalcification was carried out in 5% EDTA in 0.1 M phosphate buffer containing 4% glutaraldehyde. The solution was changed once after 24 h and decalcification was usually complete in 4 days. Both fixation and decalcification were carried out at room temperature. The tissues prepared in this way were subsequently studied as surface preparations or further processed for LM, TEM or SEM studies.

Both decalcified and undecalcified cochleae were used as whole mount prepara- tions. In both cases the specimens were dissected so the walls of the Scala tympani could be viewed along the entire extent of the basal turn; the exposed scalae were immersed in Mallory’s trichrome which stained the lining bright orange to red.

Tissue to be sectioned for light microscopy was partially dehydrated in an ethanol series (50, 70 and 95%), infiltrated with glycol methacrylate (GMA) for IO-24 h and then embedded in fresh GMA [3]. The GMA was polymerized in gelatin capsules at 45°C. 2-pm serial sections were then cut with glass knives and the sections were mounted on glass slides and stained with methylene blue-acid fuchsin. Three sets of serial sections were obtained and used to study the extent of the lining and its relation to other structures.

For SEM observations ten cochleae were dissected and then dehydrated through a graded series of acetone (30,50, 70, 80,95 and 100%). They were critical point dried, coated with gold in a vacuum evaporator and examined with an ETEC scanning electron microscope.

Tissue to be used for TEM (10 cochleae) was rinsed in 0.2 M s-collidine buffer, post-fixed for 1 h in a solution of 2% osmium tetroxide in s-collidine buffer and rinsed again in buffer. Specimens were stained en bloc in a 2% solution of uranyl acetate in 50% ethanol for 1 h, dehydrated through increasing concentrations of ethanol (70, 95 and 100%) and cleared in two changes of propylene oxide. The tissue was transferred through 30-min changes of mixtures of propylene oxide and Epon 8 12 (2 : 1, 1 : 1, 1 : 2) and undiluted resin for at least 1 h and then embedded in fresh Epon 812. Following polymerization the blocks were trimmed, oriented and affixed

25

to plastic stubs with epoxy cement or cyanoacrylate adhesive. Thin sections (40-70

nm) were cut with a diamond knife using a Sorvall MT 5000 ultramicrotome,

collected on 75 x 300 mesh copper grids or 200 mesh Formvar support grids, stained

with saturated aqueous uranyl acetate followed by lead citrate [30] and examined with a JEOL 1OOB transmission electron microscope.

Results

For descriptive purposes, the cells lining the Scala tympani of Pteronotus can be divided into three groups based on their structure and intercellular relationships: (1) those lying on the undersurface of the basilar membrane; (2) those covering the

internal surface of the otic capsule and osseous spiral lamina; and (3) those associated wtih an osmiophilic layer in the basal turn.

The cells on the undersurface of the basilar membrane had rounded or oval

shaped bodies and a complex pattern of overlapping processes (Fig. 1A). The meshwork formed by these processes was applied rather loosely to the underlying surface of the basilar membrane; through openings in the meshwork (Fig. 1) the

surface of the basilar membrane was in direct contact with the perilymph of the Scala tympani. The orientation and packing of the cells and their processes varied in

different regions of the cochlea and appeared to be random; there also appeared to be marked differences in the packing and arrangement of the cells in different individuals.

The cells covering the internal surface of the otic capsule and osseous spiral lamina were primarily arranged in a single layer but often there was considerable overlap between adjacent cells. In regions of overlap the intercellular spaces were often wide (240-270 nm) and were filled with a granular substance (Fig. 2). In

sectioned material the luminal surfaces and the nuclei of the cells appeared flattened and with the SEM neither the outlines of the cells nor the nuclei were discernible. No microvilli or other surface features were observed, but near the basilar membrane thin cytoplasmic processes similar to those on the surface of the basilar membrane were present (Fig. 1A). The cytoplasm of the cells had few organelles and was relatively homogeneous in appearance.

The cells covering the osmiophilic substance in the basal turn were dramatically

different in structure from the other cells lining the Scala tympani. The cell boundaries could not be distinguished with the SEM; with TEM the cells were

present as a single layer, but no two cells were alike in size or shape. The perilymphatic surface of each cell was relatively smooth with no microvilli or other

surface specializations; the deep surface facing the otic capsule was irregular in contour and was associated with accumulations of the material comprising the osmiophilic layer (Figs. 3 and 4). The corresponding surface of the nucleus was also irregular and its shape appeared to be influenced by the material lying adjacent to it (Figs. 2 and 3). Prominent cytoplasmic processes projected deeply into the osmiophilic material (Fig. 3).

Just beneath the cell membrane on the luminal side of the cells was a distinct

26

21

ST

Fig 2. Transmission electron micrograph of cells covering the internal surface of the otic capsule (OC).

Shown in this micrograph is the junction between two cells ivhich lie directly on the otic capsule; one cell

is clearly associated with accumulations of osmiophilic substance (OS) while the other is not. Note the

wide (240-270 nm) intercellular space (ICS) that contains the granular substance. Also, note the abrupt

termination of the osmiophilic substance; Scala tympani (ST). Calibration bar = 1 pm.

terminal web (Fig. 3) the filaments of which inserted into desmosomes along the peripheral margins of the cells. In other areas prominent arrays of filaments inserted into plaques which sometimes resembled hemidesmosomes; these were especially

numerous where the cell membrane bordered clumps of the underlying osmiophilic layer. The plaques also occurred on the cytoplasmic processes. The organelle content of the cells varied considerably from one part of the cell to the next. Prominent

rough endoplasmic reticulum was seen in many sections and Golgi saccules were also evident. In Fig. 4 the forming (immature) and the mature faces of a Golgi

apparatus can be recognized; many membrane-bound vesicles exist around the

.Fig. 1. Micrographs of the cells covering the Scala tympani surface of the basilar membrane. A. Scanning

electron micrograph showing a bulging nucleus (N) and numerous cytoplasmic processes (P) radiating

from cell bodies to form a meshlike network. Note that similar processes (arrowheads) are also present

adjacent to the edges of the basilar membrane. B. Transmission electron micrograph of the pars tecta. Note the nucleus (N) of a cell on the undersurface of the basilar membrane, the thin cytoplasmic

processes (P) and the holes (arrows) in the mesh-like arrangement of the processes where the basilar

membrane is directly exposed to the perilymph in the Scala tympani (ST). The thickening of the pars tecta

represents a remnant of an embryonic blood vessel and some of the internal structure of the vessel is still

evident. In A, the calibration bar represents 10 pm and in B, 1.0 pm.

Fig. 3. Transmission electron micrograph showing some of the features of a cell associated with the

osmiophilic layer. Note the smooth, flat surface of the nucleus adjacent to the scala tympani and the

irregular contour of the surface facing the inner wall of the otic capsule. Also shown are numerous

cytoplasmic processes, some of which appear to be degenerating (DP). The extracellular osmiophilic

substance is marked by the black star; an open star marks what appears to be an intracellular

accumulation of the same substance. Also seen in this micrograph is the terminal web (TW). Calibration

bar = 1 pm.

Fig. 4. The organelles and internal structure of a cell associated with accumulations of osmiophilic substance. This section shows a prominent Golgi complex (G); the mature face is assoeiated with many

vesicles (V) filled with a granular material. Near the deep surface of the cell, the granular material

becomes condensed and it is similar in appearance to the substance accumulated below the cell; Scala

tympani (ST). Calibration bar = 0.5 pm.

29

mature face and between the Golgi apparatus and the deep surface of the cells. The membrane-bound vesicles closest to the cell membrane contained an osmiophilic

granular substance similar in appearance to the material which accumulated deep to

the cells. The micrographs strongly suggest that the Golgi complex of these cells synthesizes the substance that abounds deep to the cell surface and that the substance may be transported by carrier vesicles and released by exocytosis at the

cell membrane. In many micrographs there were also indications that the osmiophilic substance could accumulate within the cytoplasm without being membrane bound.

The cytoplasmic processes which projected deep into the osmiophilic layer were long and thin in profile. The membranes of these processes were usually distinct near the cell body but they, and the processes themselves, often became indistinct within

the osmiophilic layer and appeared to be undergoing degeneration (Fig. 3). The compact osmiophilic material which accumulated between the epithelium and

underlying bone stopped abruptly near the edge of the basilar membrane and it

extended from the regions of the round window and cochlear aqueduct openings approximately 5 mm along the ca. 12 mm length of the basilar membrane. The

junction between the cells which appear to produce the osmiophilic layer and those

which do not, was distinct and abrupt (Fig. 2). The osmiophilic layer was situated

directly on the basal lamina (Fig. 2). An extensive search of serial sections revealed no blood vessels in or near the osmiophilic layer; all vessels were deep and confined to the underlying bone. In the freshly killed animal the osmiophilic layer was

transparent with a slightly yellowish tint and was easily distinguished from accumu- lations of red blood cells.

Discussion

The cells lining the perilymphatic fluid-filled spaces of the cochlea were described in the last century by Claudius [6], Hensen [9], Boettcher [4] and Retzius [26]. More

recently, their ultrastructure has been studied in the rat by Iurato [17], in the guinea

pig by Lim [ 181, Angelborg and Engstrom [2] and in the chinchilla by Franke [8]. In general these investigators have reported that a thin layer of mesothelial cells lines

the undersurface of the basilar membrane and the adjacent Scala tympani surface of the otic capsule. This layer is thinnest under the basilar membrane. The cells are oval with long branching processes and they are most numerous in the basal turn where

they are oriented longitudinally; in the upper turns there are also radially oriented cells. The long thin processes of the cells overlap each other extensively but leave many holes so that the basilar membrane is directly in contact with the perilymph [5,18,27]. The basic description of the cells in other animals is also true for the cells in the non-specialized region (second and apical turns) of the cochlea of Pteronotus and all other species of bats which we have examined (Macrotus, Artibeus, Tadarida, Mormoops). There is, however, some variation in the number of layers in different animals. In Pteronotus and guinea pigs [2,18] there is usually only a single layer of cells. In the chinchilla Franke [8] found from two to five cell layers and Angelborg and Engstrom [2] reported that in the squirrel monkey there were many layers. They

also noted the presence of a single kinocilium on the perilymphatic surface of each cell in the guinea pig; in Pteronotus no kinocilia were observed.

The function of the cells lining the Scala tympani is not known, but it has been shown that the cells are highly active in the synthesis of RNA [ 191 and also that they are highly phagocytic [ 1,7]. Except for Rye [24,25], no other investigators have found that the cells lining the Scala tympani in any part of the cochlea, in any animal, are associated with the build-up of an osmiophilic or other type of substance. Like Rye7

we have not encountered this substance in any other species of Pteronotus or any other genus of Microchiroptera. The present investigation on the specialized lining in

Pteronotus has focused on the cells and their ultrastructure, while Rye’s studies were

concerned primarily with the nature of the accumulated substance. The results and

interpretations of the present study differ significantly from hers. On the basis of TEM micrographs and a hemoglobin stain (Buffalo black) Rye concluded that the

substance of the thickened lining was lysed red blood cells and massed hemoglobin.

She also made the observation that the thick substance was absent in fetal and

young animals. This observation, in conjunction with the known phagocytic ability of these cells [l] might lead to the conclusion that the thick osmiophilic layer simply

represents the buildup of debris from phagocytized red blood cells and other foreign material. Thus, this thick layer would represent the product of a protective system which keeps the perilymph from being contaminated. On the other hand, it seems evident from TEM micrographs obtained in this investigation that the osmiophilic

substance which accumulates in and around these cells is not a product of phago- cytosis but a product of the Golgi apparatus of the cells themselves.

The fact that the thick osmiophilic substance is unique to Pteronotus parneliii

suggests that it might in some way be related to their unusual hearing capacities. One of the most unusual aspects of their ear is the remarkable resonance of the

system at or near 61 kHz, the frequency to which the ear is sharply tuned. When the ear is stimulated with brief tone pulses at or near this frequency, CM potentials

persist well after .the end of the stimulus; they constitute what Suga and his colleagues [28,29] have called the CM-aft, the frequency of which is the resonance frequency of the ear. Recent experiments by Schuller, Vater and Henson (unpub-

lished) have indicated that when the ear of Pteronotus is stimulated by sound near the resonance frequency of the animal’s ear, there may be strong co&ear re-emis- sions (Kemp echoes). It is tempting to correlate the enlarged tympanic scalae of Pteronotw with these unusual resonance properties. The possibility exists that the total volume of the scalae and the mechanical properties of the system might be determined to some extent by the osmiophilic substance that appears to be produced by the cells lining the Scala tympani. Another possibility is that the thick lining may help to reduce energy reverberations within the cochlea by the creation of soft rather than hard bony walls. In any case, the conditions in Pteronotus raise the question as to how and to what extent the size and nature of the perilymphatic fluid-filled scalae influence the acoustic properties of the system.

31

Acknowledgements

We would like to thank Janice Aydlette photographic plates. This investigation was Health Service, NS 12445.

References

for assistance with the preparation of supported by a grant from the Public

1 Angelborg, C. (1974): Distribution of macromolecular tracer particles (Thorotrast) in the cochlea. An

electron microscopic study in guinea pig. Acta Otolaryngol. Suppl. 319, 19-41.

2 Angelborg, C. and Engstrom, B. (1974): The tympanic covering layer. An electron microscopic study

in guinea pig. Acta Otolaryngol. Suppl. 319, 43-56.

3 Bennett, H.S., Wyrick, A.D., Lee, SW. and McNeil, J.H. Jr. (1976): Science and art in preparing

tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate. glass

knives and simple stains. Stain Technol. 5 1, 7 I-97.

4 Boettcher, A. (1869): Uber Entwickhmg und Bau des Gehorlabyrinths nach Untersuchungen an

Saugethieren. Nova Acta Academic Naturae curiosorum 35, I-203.

5 Bredberg, G., Ades, H.W. and Engstrom. H. (1972): Scanning electron microscopy of the normal and

pathologically altered organ of Corti. Acta Otolaryngol. Suppl. 301, 3-48.

6 Claudius, M. (1856): Bemerkungen tiber den Bau der hautigen Spiralleiste der Schnecke. Z. Wiss.

Zool. 7, 154-161.

7 Duvall, A.J. and Sutherland, C.R. (1970): The ultrastructure of the extrasensory cells in the cochlear

duct. In: Biochemical Mechanisms in Hearing and Deafness. pp. 1499170. Editor: M.M. Papareila.

Charles C. Thomas, Springfield, Ill.

8 Franke, K. (1979): Fine structure of the tissue lining the cochlear perilymphatic space against the

bony labyrinthine capsule. Arch. Otorhinolaryngol. 222, 16 l- 167.

9 Hensen, V. (1863): Zur Morphologic der Schnecke des Menschen und der Saugethiere. Z. Wiss. Zool.

13, 481-512.

IO Henson, M.M. (1973): Unusual nerve-fiber distribution in the cochlea of the bat. P~eronorus p.

parnellii (Gray). J. Acoust. Sot. Am. 53, 1739-1740.

I I Henson, M.M. (1978): The basilar membrane of the bat, Preronorus p. pamellir. Am. J. Anat. 153.

143-157.

12 Henson. M.M. (1979): The lining of the Scala tympani in the bat, Pteronotur p. pamdii. Anat. Rec.

193, 744.

I3 Henson. M.M. and Henson, O.W. Jr. (1979): The cochleae of ‘constant frequency’ bats: model

systems for studying features associated with fine frequency analysis. In: Abstracts of the Second

Midwinter Research Meeting, Association for Research in Otolaryngology, p. 32. Editor: D.J. Lim.

I4 Henson, M.M., Henson, O.W. Jr. and Goldman, L.J. (1977): The perilymphatic spaces in the cochlea

of Pteronotur p. pamellii. Anat. Rec. 187. 767.

I5 Henson, M.M. and Jenkins, D.B. (1981): The Scala tympani and the ultrastructure of the tympanic

covering layer in the bat, PteronorusparneNiipamel/ii. In: Abstracts of the Fourth Midwinter Research

Meeting, Association for Research in Otolaryngology, p. 84. Editor: D.J. Lim.

I6 Henson, M.M., Jenkins, D.B. and Henson, O.W. Jr. (1982): The cells of Boettcher in the bat,

Pteronotur p. pnmellii. Hearing Res. 7, 91- 103.

I7 lurato, S. (1962): Submicroscopic structure of the membranous labyrinth. III. The supporting

structure of Gxti’s organ (basilar membrane, limbus spirale and spiral ligament). Z. Zellforsch.

Mikros. Anat. 56, 49-96.

18 Lim, D. (1970): Surface ultrastructure of the cochlear perilymphatic space. J. Laryngol. Otol. 84,

413-428.

I9 L&e, P. (1974): Autoradiographische Untersuchungen am lnnenohr nach cochlearer Perfusion mit

‘H-Uridin. Arch. Oto-Rhino-Laryngol. 208, 61-70.

32

20 Pollak, G.D., Henson, O.W. Jr. and Johnson, R. (1979): Multiple specializations in the peripheral

auditory system of the CF-FM bat, Pteronotw pamellii. J. Comp. Physiol. 131, 2555266.

21 Pollak, G.D., Henson, O.W. Jr. and Novick, A. (1972): Cochlear microphonic audiograms in the ‘pure

tone’ bat, Chilonycteris parnellii parnellii. Science 176, 66-68.

22 Pye, A. (1967): The structure of the cochlea in Chiroptera. III Microchiroptera: Phyllostomatoidea. J.

Morphol. 121, 241-254.

23 Pye, A. (1978): Aspects of cochlea structure and function in bats. In: Proceedings of the Fourth

International Bat Research Conference, pp. 73-83. Editors: R.J. Olembo, J.B. Castelino and F.A.

Mutere. Kenya National Academy for Advancement of Arts and Sciences, Kenya Literature Bureau.

24 Pye, A. (1980): The structure of the cochlea in some new world bats. In: Proceedings of the 5th

International Bat Research Conference, pp. 39-49. Editors: D.E. Wilson and A.L. Gardner. Texas

Tech. Press, Lubbock, Texas.

25 Pye, A. (1980): The cochlea in Pteronotw pamellii. In: Animal Sonar Systems, pp. 965-967. Editors:

R.-G. Busnel and J.F. Fish. Plenum Press, New York.

26 Retzius, G. (1884): Das Gehororgan der Wirbelthiere. II. Das Gehijrorgan der Reptilien, der Vogel

und der Saugethiere. Samson and Wallin, Stockholm.

27 Spoendlin, H. (1966): The organization of the cochlear receptor. Bibl. Oto-Rhino-Laryngol. 13, l-227.

28 Suga, N. and Jen, P.H.S. (1977): Further studies on the peripheral auditory system of ‘CF-FM’ bats

specialized for fine frequency analysis of Doppler-shifted echoes. J. Exp. Bid. 69, 207-232.

29 Suga, N., Simmons, J.A. and Jen, P.H.S. (1975): Peripheral specialization for fine analysis of

Doppler-shifted echoes in the auditory system of the ‘CF-FM’ bat, Pteronotus pnrnellii. J. Exp. Biol.

63, 161-192.

30 Venable, J.H. and Coggeshall, R. (1965): A simplified lead citrate stain for use in electron microscopy.

J. Cell Biol. 25, 407-408.