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The cells of Boettcher in the bat, Pteronotus p. parnellii
Hearing Research, 7 ( 1982) 9 1- 103 Elsevier Biomedical Press
91
The cells of Boettcher in the bat, Pteronotus p. parnellii M.M. Henson I, D.B. Jenkins 2 and O.W. Henson, Jr. 2 Deportments
of ’ Surgery ond ’ Anatomy The University of North Corolino, School of Medicine, Hill, NC, 27514, U.S.A. (Received
14 October
1981; accepted
21 December
Chapel
1981)
The structural characteristics, distribution and intercellular relationships of the cells of Boettcher were studied in the mustache bat, Preronofus p. pornellii. The cells of Boettcher have many structural features similar to those described in other mammals,but in Pteronofw they are distributed throughout the cochlea and are associated with relatively large amounts of secretory and/or absorptive material. Much of this material seems IO be derived from or contribute to, a darkly staining upper layer of the basilar membrane. This material accumulates in elaborate microvillus-filled intercellular channels which are restricted to an area near the basilar membrane. The channels communicate with the basilar membrane surface through wide intercellular spaces and through small canals. The microvillus-filled channels are confluent with large extracellular spaces between Boettcher’s cells and a single row of cells which form the floor of the outer tunnel. The latter have irregular shaped nuclei, contain many vacuoles and like Boettcher’s cells, are associated with huge amounts of basilar membrane-like material. Observations on Pleronotus. as well as other species of bats, do not support concepts relating Boettcher’s cells to hair cell innervation patterns or to high frequency hearing. Key words:
organ
of Corti;
bat; hearing.
Introduction The cells of Boettcher are a localized group of cells in the organ of Corti and they are named in recognition of the man who described them more than 100 years ago. Descriptions of these cells have been limited to a few mammals, namely cat [2,5,15], guinea pig, squirrel monkey, man [ 151 and the bat, Myoris 1ucifugu.s [ 161. In general it has been found that the cells of Boettcher: (1) are restricted to the basal portion of the cochlea; (2) lie in rows directly on the vestibular surface of the basilar membrane; (3) have a dense, organelle-filled cytoplasm which distinguishes them from all surrounding cells; and (4) have elaborate interdigitating surface membranes where they border each other near the basilar membrane. Histochemical studies have also established that these cells have high acid phosphatase activity [14] and nonspecific esterases and proteinases [3,6]. These findings and the presence of numerous free ribosomes in the cytoplasm have led to speculations that the cells are secretory and/or absorptive in function; however, there have been no clues as to the 0378-5955/82/0000-0000/$02.75
0 1982 Elsevier
Biomedical
Press
92
type of material secreted or absorbed. In addition, the restricted position of the cells in the basal turn of the cochlea has led to the suggestion that they may be associated with high frequency hearing [ 151. For the past 15 years our laboratory has been particularly concerned with anatomical, behavioral and physiological aspects of ultrasonic hearing in the mustache bat, Pteronotus p. parnellii. This species is interesting from an acoustic point of view in that their sense of hearing is sharply tuned to narrow frequency bands which correspond to constant frequency components in their biosonar signals [7,17,18,20,2 1] and it seems likely that the sharp tuning and hearing properties are to a large degree established by the anatomical attributes of the cochlea. The basilar membrane, for example, has well developed thickenings (9,191 and it has been shown that the pattern of change in the width of the membrane from base to apex is different from that in other bats and other mammals [9]; attention has also been directed to an unusual nerve fiber distribution in the basal turn [8]. to supporting cells in and around the outer tunnel (fourth space of Nuel) [lo] and to dilations and constrictions of the fluid filled spaces [ 121. The purpose of this report is to describe certain features of the cells of Boettcher in Pteronotus which are different from those known to occur in other mammals. We will also show that these cells have a consistent relationship to large supportive cells forming the floor of the outer tunnel and to extracellular spaces and secretory and/or absorptive material which accu: mulates in these spaces.
Materials and Methods Mustache bats, Pteronotusparnelliiparnellii, from Jamaica, W.I., were used in this study. Animals were decapitated, the head cut in the midsaggittal plane and the cochleae rapidly removed. The cochleae were immersed in fixative consisting of 4% glutaraldehyde and 4% sucrose in 0.2 M s-collidine buffer, pH 7.3. The apex of each cochlea was opened and. the round window membrane was ruptured 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 four days. Both fixation and decalcification were carried out at room temperature, and the tissues prepared in this way were subsequently studied as surface preparations or further processed for LM, TEM or SEM studies, For nuclear staining the entire cochlea was carefully dissected to remove overlying bone and tissue was then stained with gallocyanin [13]. After staining. the different turns and the hook region were isolated and the specimens were dehydrated in an ethanol series, placed in xylene and then mounted on a slide with DPX and coverslipped. With the light microscope the different cellular elements of the organ of Corti could be easily recognized on the basis of their position and organization in rows. In order to obtain information on the population density and distribution of Boettcher’s cells, the images of the nuclei were projected onto paper with the aid of a drawing tube and a montage of the nuclei within the spiraling organ of Corti was
93
constructed; nuclear densities were counted per 0.1 mm length from the hook to the apex. Montages and counts were made from the different regions in six cochleae. Tissue to be sectioned for light microscopy was partially dehydrated in an ethanol series of 50% 70% and 95% infiltrated with glycol methacrylate (GMA) for lo-24 h and then embedded in fresh GMA [I]. 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 cochleae, cut in different planes, were processed in this way and the material was used to assess intercellular relationships and light microscopic features. For SEM observations of the cells of Boettcher the overlying Hensen’s cells were removed from the fixed material by treating the tissue with 1% trypsin for various periods of time [IO]. The tissue was then dehydrated with a graded series of acetone, critical point dried, coated with gold and examined with an ETEC scanning electron microscope. Tissue to be used for TEM was rinsed in 0.2 M s-collidine buffer, postfixed 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 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 cut, oriented and affixed 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 and lead citrate [22] and examined with a Joel 1OOB transmission electron microscope. The following descriptions of ultrastructural details are based on micrographs obtained from all turns of the cochleae.
Results The relationships of the cells of Boettcher to other cells in the organ of Corti of Pleronorus are shown schematically in Fig. 1. The cells are situated on the surface of the basilar membrane at or near the junction of the pars pectinata and the spiral ligament. They are bounded on the side toward the modiolus by a single row of cells which are morphologically distinct from all others in the organ of Corti; in accordance with their position these cells will be referred to as the basal cells of the outer tunnel or simply tunnel floor cells. On the side toward the spiral ligament are the cells of Claudius and above Boettcher’s cells are the cells of Hensen. These intercellular relationships are maintained throughout the cochlea. In this report only the relationships of Boettcher’s cells to each other, to the basilar membrane and to the basal cells of the outer tunnel will be considered. After trypsin digestion of the overlying tissue, the cells of Boettcher were observed in SEM micrographs as a continuous cord of cells’(Fig. 2A). Along the
94
Pig. 1. Schematic diagram of cellular elements associated with the outer tunnel of the organ of Corti in Pfero!rofusp. putwellii. Illustrated are the relationships of Boettcher’s cells (B) to the pars pectinata of the basilar membrane (PP). the outer tunnel (OT). the basal cells of the outer tunnel (BOT). the cells of Deiters (D). the cells of Hensen (H). and the spiral ligament (SL). Also shown are several outer hair cells (OHC) and the cell which forms the roof (tectum) of the outer tunnel (TOT).
inside edge of the cord (Fig. 2A) were the nuclei of the tunnel floor cells and in places where these were removed (Fig. 2B) there were irregular accumulations of material interposed between the cells of Boettcher and the adjacent row of Deiters’ cells. From TEM and LM micrographs it was determined that the cells of Boettcher had spherical nuclei 4-5 pm in diameter. Although irregular in shape, the cells themselves measured approximately 6-8 pm in width and 6-7 pm in height (Fig. 3). TEM micrographs showed the same general characteristics that have been described for other species. This included large numbers of microtubules throughout the cytoplasm (Fig. 4); both bundles and individual tubules appeared to be randomly oriented but occasionahy a bundle was seen encircling the nucleus, as described by Kimura (161. The diameter of these tubules, whether in bundles or isolated was approximately 25 nm. The cytoplasm also contained mitochondria, lysosomes, a Golgi apparatus, vacuoles, vesiculated bodies and lipofuchsin granules; many free ribosomes were scattered throughout the cytoplasm. These ultrastructural features can be seen in several of the micrographs (Figs. 4-7) included in this report. In sharp contrast to the dense cytoplasm of Boettcher’s cells, the cytoplasm in all adjacent cells was almost completely devoid of any organelles (Fig. 3A). The cytoplasm (Figs. 3B and 5) and the spaces around the tunnel floor cells (Fig. 7) were crowded with clear, irregularly shaped vesicles and vacuoles of various sizes. Some of these appeared to be membrane bound, but the exact boundaries of the cells and vacuoles were difficult to assess. The cytoplasmic organelles of the tunnel floor cells included lysosomes and mitochondria and almost all of these were crowded into the basal portion of the cells. The nuclei were very irregular in shape and the shape seemed to. be influenced to a large degree by the pressure applied by surrounding vacuoles; however, deep indentations were also observed (Fig. 5). The average
Fig. 2. SEM micrographs of the ceils of Boettcher in the basal turn. These micrographs were obtained by disrupting and removing overlying tissue with trypsin. In A note the cord-like appearance of the cells of Boettcher (B) and their bulging nuclei (arrows); the nuclei of the adjacent tunnel floor cells are also evident (arrowheads). 1320 X In B some of the material which accumulates along the inner side of the cord of Boettcher’s cells can be seen next to the third row of Deiters’ cells (D). 3300X.
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Fig. 3. TEM micrographs of the cells of Boettcher. A shows a section transverse to the long axis of the cord. Note the relationship of three Boettcher’s cells (B) to each other and to the basilar membrane (BM) (beginning of the second turn). The arrow points to the thin osmophilic layer of material that underlies Boettcher’s cells and the tunnel floor cells. 6600X. B shows a longitudinal section along the cord of Boettcher’s cells in the apical turn. 5700X.
Fig. 4. Illustrations of the junctions between adjacent Boettcher’s cells. In A the types of junctions that occur extensively in the nuclear and supranuclear portions of each cell are evident. 19000X. B shows an example of the elaborate, microvillus-filled channels that occur near the bases of the cells. Note the dark material (M) which lies within the channel and the numerous microtubules within the cytoplasm of the cells (apical turn). 24000X.
Fig. 5. Microvillus-filled channels and their relationship to extracellular spaces near the tunnel floor cells. A shows a section parallel to the basilar membrane through the channels and illustrates the connections (arrow) of the channels with the extracellular spaces filled with darkly staining material. The clear spaces with vesicles represent the bases of the tunnel floor cells. 20000X. B shows a section at a slightly higher level in the cord. Note the deep cleft in the nucleus of the tunnel floor cell and the large clear vesicles (apical turn). 8000X.
Fig. 6. TEM micrographs of pore-like canals that exist between the microvillus-filled channels (C) and the basilar membrane (BM). A shows a longitudinal section through a canal containing the basilar membranelike material (basal turn). Note the many microtubules in this micrograph. 24650X. B is a transverse section through several channels and canals in the second turn. Continuity of the material with the upper layer of the basilar membrane can be seen. 23 250 X .
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diameter of the nuclei was about 7pm and thus they were significantly larger than the nuclei of Boettcher’s cells. Because of their size and distorted appearance the nuclei of the tunnel floor cells could be easily distinguished from those of the other cells when examined in whole mount preparations stained with the nuclear stain, gallocyanin. From such preparations, as well as serial sections, it was observed that both the tunnel floor cells and Boettcher’s cells occurred throughout the cochlea. The density of the tunnel floor cells was constant per unit length from the beginning of the hook to the apex, with about 10 cells per 100 pm. The density of Boettcher’s cells changed slightly. There were only two rows of cells in the hook region resulting in 20-30 cells per 100 pm; the number gradually increased to 35-40 cells per 100 pm and this was the number found throughout most of the remainder of the cochlea. There were, however, two regions with approximately 50 cells per 100 pm; these were from the middle to the distal end of the sparsely innervated region [8] and at the apex. The structure of the intercellular junctions between adjacent Boettcher’s cells varied in different levels of the cord. Junctions adjacent to the nucleus (Fig. 4A) and at supranuclear levels were narrow. The intercellular junctions below the level of the
Fig. 7. Transverse section through the extracellular space between a Boettcher’s cell (B) and the basal cell of the outer tunnel (BOT’) in the hook region. Note the elongate microvilli protruding into the vesicle-filled space. This section is near the point of continuity of a microvillus-filled channel and the extracellular space. 15 000 X
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nucleus had numerous interdigitating microvilli (Fig. 4B). Sections through the lower part of the cord of Boettcher’s cells showed that the spaces between interdigitating microvillus surfaces were actually channels between adjacent cells. The channels were continuous with extracellular spaces between Boettcher’s cells and the tunnel floor cells (Fig. 5A). These spaces were always filled with a dark (osmophilic) substance that had the same staining properties as the layer of the basilar membrane on which the cells rested (Fig. 5A). This layer may represent a thick basal lamina (see Fig. 3A). When sections were cut through the cord at slightly higher levels, the darkly staining material was observed to lie between adjacent tunnel floor cells; this material apparently corresponds to the irregularly shaped clumps of material observed in SEM micrographs (Fig. 2). Sections cut parallel and perpendicular to the surface of the basilar membrane showed that there were not only wide intercellular spaces through which the basilar membrane material projected, but also that there were pore-like canals. The continuity of these small canals with the larger microvillus-filled channels was evident when sections were cut both transverse and parallel to the longitudinal axis of the microvillus-filled channels (Fig. 6). At the point of confluence of the microvillus-filled channels with the extracellular spaces many microvilli were seen to project freely into the extracellular spaces. The micrograph in Fig. 7 shows some of these microvilli and vacuole-like profiles that were often encountered in the extracellular space, in the tunnel floor cell cytoplasm and to a limited extent in the microvillus-filled channels.
Discussion
The present study provides new information about the cells of Boettcher which relates to: (1) their distribution and density; (2) the presence of contiguous intercellular channels and large extracellular spaces; (3) the existence of several different types of secretory or absorptive material within the channels and spaces; (4) a consistent relationship of basilar membrane material to the channels; and (5) a consistent relationship to an unusual cell type which forms the floor of the outer tunnel. The concept of a distinct cell type forming the floor of the outer tunnel was first advanced by Henson and Henson [ 111. Although more details concerning these cells will be forthcoming, it is necessary here to note their close association with the cord of Boettcher’s cells and with the large extracellular spaces that exist between these two cell types. Both cells exist side by side throughout the cochlea and seem to be associated with the same types of extracellular material. It is not known if tunnel floor cells are present in mammals other than bats or if they, or Boettcher’s cells, exist in isolation. The distribution and density of Boettcher’s cells in Pteronotus is markedly different from that in other mammals. In other groups where Boettcher’s cells have been described they have been limited to the basal turn. In other bats which we have examined (Tuduridu, Mormoops, Rhinolophus) the distribution of Boettcher’s cells and tunnel floor cells was similar to that in Pteronorus and, therefore, may be a
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characteristic feature of Microchiroptera rather than a feature unique to Pteronotus. Although the cells extend throughout the cochlea in the bats thus far examined, the number of rows is considerably less than in the cat where there may be up to 17 rows and in the squirrel monkey which has up to 20 rows in certain regions [ 151. In our study of the population density of these cells we were interested in determining whether there were regions where the density changed and if so, could they be correlated with the unusual hearing capacities of Pteronorus, the innervation pattern and/or the marked volumetric differences known to occur in the Scala tympani and Scala vestibuli. The relatively constant density and distribution of Boettcher’s cells, however, argues against a direct correlation with any known variations in these features. Ishiyama et al. [15] have suggested that the function and distribution of Boettcher’s cells might be related to the efferent endings and the products they release into the fluid-filled spaces of Nuel. Such endings are numerous in the basal turn and decrease toward the apex. The data obtained from Pteronotus show nothing to dispute such an association; however the European horseshoe bat, Rhinolophus ferrumequinum, has no efferent fibers to the outer hair cells [4] yet the cells of Boettcher are distributed throughout the cochlea. The microvillus-filled channels that are described in this report were only recognizable as channels when sections were made along the axis of the channels (Figs. 4 and 5A) or when the channels were transected (Figs. 6 and 7). Other investigators [5,15,16] have noted numerous interdigitating microvilli between adjacent Boettcher’s cells and projections of small amounts of basilar membrane material, but the existence of microvillus-filled channels which communicate extensively with the underlying surface of the basilar membrane and with other extracellular spaces has not previously been described. Their presence in other mammals, however, is strongly suggested by published micrographs [5,15,16]. The intercellular spaces between the bases of adjacent Boettcher’s cells, the numerous small channels and the presence of intercellular vesicles strongly support the suggestion of Ishiyama et al. [15] that the cells have an important absorptive function; it would appear that whatever is absorbed must pass through the basilar membrane or be part of it. It is interesting to note that if the entire basilar membrane surface of Boettcher’s cells were covered with microvilli, then the cord of cells could not be firmly’anchored to the vibrating surface. In this case the biological solution seems to be the creation of the porous surface which permits the material to collect in the microvillus-filled channels. This arrangement appears to be unique to the organ of Corti. We do not know the nature of the material that may be secreted or absorbed by the cells of Boettcher, but there is a close association with a darkly staining (osmophilic) substance that is clearly a constituent of the deepest layer of the basilar membrane. The cells are also associated with clear, vesicle-like material. It may be that the cells of Boettcher are associated with the maintenance and turnover of the basilar membrane. Perhaps there is a constituent of the membrane that occurs only in specific parts of the cochlea in many mammals, and is important in governing the vibratory properties of the system.
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Acknowledgement This work was supported by USPHS Grant 12445. References 1 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, 71-97. 2 Boettcher, A. (1869): Ueber Entwicklung und Bau des Gehorlabyrinths nach Untersuchungen an Saugetieren. Novorum Actorum Academiae Caesareae Leopoldino-Carolinae Germanicae Naturae Curiosorum 35. I-203. 3 Brodmann, G. and Giebel, W. (1978): Histochemische Untersuchungen zur Funktion und Verteilung der Boettcherschen Zellen in der Goldhamstercochlea. Arch. Oto-Rhino-Laryngol. 220, IOS- I 16. 4 Bruns, V. and Schmieszek, E. (1980): Cochlear innervation in the greater horseshoe bat: Demonstration of an acoustic fovea. Hearing Res. 3.27-43. 5 Duvall, A.J. 111 and Sutherland, C.R. (1970): The ultrastructure of the extrasensory cells in the cochlear duct. In: Biochemical Mechanisms in Hearing and Deafness, pp. 149-170. Editor: M.M. Paparella. Charles C. Thomas, Springfield, 111. 6 Giebel. W. and Brodmann, G; (1977): Histochemie der Esterasen und Endopeptidasen im Innenohr. Arch. Oto-Rhino-Laryngol. 216, 521-522. 7 Grinnell. A.D. (1967): Mechanisms of overcoming interference in echolocating animals. In: Les Systems Sonars Animaux Biologie et Bionique Tome I, pp. 451-480. Editor: R.G. Busnel. Laboratoire de Physiologic Acoustique INRA-CNRZ, Jouy-en-Josas 78, France. 8 Henson. M.M. (1973): Unusual nerve-fiber distribution in the cochlea of the bat, Pteronorur p. purnellii (Gray). J. Acoust. Sot. Am. 53. 1739-1740. 9 Henson, M.M. (1978): The basilar membrane of the bat, Preronotm p. purnellii. Am. J. Anat. 153, 143-157. 10 Henson. M.M. and Henson, O.W. Jr. (1979): Some aspects of structural organization in the cochlea of the bat. Pferonorus p. purnellii. Scanning Electron Microsc. 1979/111. 975-982. I I Henson. M.M. and Henson, O.W. Jr. (1979): The supportive elements of the organ of Corti in the bat, Pteronorus p. purneilii. Presented at the Meeting of the Southern Society of Anatomists, Washington, D.C. Anat. Rec. (in press). 12 Henson, M.M. and Jenkins. D.B. (1981): The Scala tympani and the ultrastructure of the tympanic covering layer in the bat. Pteronotus porneliii pumellii. Abstracts of the Fourth Midwinter Research Meeting. Association for Research in Otolaryngology. p. 33. I3 Humason. G.L. (I 972): Animal Tissue Techniques. W.H. Freeman and Company, San Francisco. I4 Ishii. T. and Balogh, K. Jr. (1966): Acid phosphatase activity in the inner ear. Acta Oto-laryngol. 62, 185-192. 15 Ishiyama, E., Cutt, R.A. and Keels, E.W. (1970): Distribution and ultrastructure of the Boettcher’s cells in mammals. Ann. Otol. RhinoI. Laryngol. 79, 54-69. I6 Kimura. R.S. (1975): The ultrastructure of the organ of Corti. Int. Rev. Cytol. 42, 173-222. I7 Pollak. G.D.. Henson, O.W. Jr. and Johnson, R. (1979): Multiple specializations in the peripheral auditory system of the CF-FM bat, Pteronorus purnellii. J. Comp. Physiol. 131. 255-266. I8 Pollak. G.D., Henson. O.W. Jr. and Novick, A. (I 972): Cochlear microphonic audiograms in the ‘pure tone’ bat. Chilotaycteris purnellii purnellii. Science 176. 66-68. I9 Pye, A. (1966):The structure of the cochlea in Chiroptera. I. Microchiroptera: Emballonuroidea and Rhinolophoidea. J. Morphol. I 18. 495-S IO. 20 Suga, N. and Jen, P.H.S. (I 977): Further studies on the peripheral auditory system of ‘CM-FM’ bats specialized for fine frequency analysis of Doppler-shifted echoes. J. Exp. Biol. 69, 207-232. 21 Suga, N.. Simmons, J.A. and Jen, P.H.S. (1975): Peripheral specialization for fine analysis of Doppler-shifted echoes in ‘CF-FM’ bat, Preronorus pumellii. J. Exp. Biol. 63. I6 I - 192. 22 Venable. J.H. and Coggeshall. R. (1965):A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25. 407-408.
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The cells of Boettcher in the bat, Pteronotus p. parnellii M.M. Henson I, D.B. Jenkins 2 and O.W. Henson, Jr. 2 Deportments
of ’ Surgery ond ’ Anatomy The University of North Corolino, School of Medicine, Hill, NC, 27514, U.S.A. (Received
14 October
1981; accepted
21 December
Chapel
1981)
The structural characteristics, distribution and intercellular relationships of the cells of Boettcher were studied in the mustache bat, Preronofus p. pornellii. The cells of Boettcher have many structural features similar to those described in other mammals,but in Pteronofw they are distributed throughout the cochlea and are associated with relatively large amounts of secretory and/or absorptive material. Much of this material seems IO be derived from or contribute to, a darkly staining upper layer of the basilar membrane. This material accumulates in elaborate microvillus-filled intercellular channels which are restricted to an area near the basilar membrane. The channels communicate with the basilar membrane surface through wide intercellular spaces and through small canals. The microvillus-filled channels are confluent with large extracellular spaces between Boettcher’s cells and a single row of cells which form the floor of the outer tunnel. The latter have irregular shaped nuclei, contain many vacuoles and like Boettcher’s cells, are associated with huge amounts of basilar membrane-like material. Observations on Pleronotus. as well as other species of bats, do not support concepts relating Boettcher’s cells to hair cell innervation patterns or to high frequency hearing. Key words:
organ
of Corti;
bat; hearing.
Introduction The cells of Boettcher are a localized group of cells in the organ of Corti and they are named in recognition of the man who described them more than 100 years ago. Descriptions of these cells have been limited to a few mammals, namely cat [2,5,15], guinea pig, squirrel monkey, man [ 151 and the bat, Myoris 1ucifugu.s [ 161. In general it has been found that the cells of Boettcher: (1) are restricted to the basal portion of the cochlea; (2) lie in rows directly on the vestibular surface of the basilar membrane; (3) have a dense, organelle-filled cytoplasm which distinguishes them from all surrounding cells; and (4) have elaborate interdigitating surface membranes where they border each other near the basilar membrane. Histochemical studies have also established that these cells have high acid phosphatase activity [14] and nonspecific esterases and proteinases [3,6]. These findings and the presence of numerous free ribosomes in the cytoplasm have led to speculations that the cells are secretory and/or absorptive in function; however, there have been no clues as to the 0378-5955/82/0000-0000/$02.75
0 1982 Elsevier
Biomedical
Press
92
type of material secreted or absorbed. In addition, the restricted position of the cells in the basal turn of the cochlea has led to the suggestion that they may be associated with high frequency hearing [ 151. For the past 15 years our laboratory has been particularly concerned with anatomical, behavioral and physiological aspects of ultrasonic hearing in the mustache bat, Pteronotus p. parnellii. This species is interesting from an acoustic point of view in that their sense of hearing is sharply tuned to narrow frequency bands which correspond to constant frequency components in their biosonar signals [7,17,18,20,2 1] and it seems likely that the sharp tuning and hearing properties are to a large degree established by the anatomical attributes of the cochlea. The basilar membrane, for example, has well developed thickenings (9,191 and it has been shown that the pattern of change in the width of the membrane from base to apex is different from that in other bats and other mammals [9]; attention has also been directed to an unusual nerve fiber distribution in the basal turn [8]. to supporting cells in and around the outer tunnel (fourth space of Nuel) [lo] and to dilations and constrictions of the fluid filled spaces [ 121. The purpose of this report is to describe certain features of the cells of Boettcher in Pteronotus which are different from those known to occur in other mammals. We will also show that these cells have a consistent relationship to large supportive cells forming the floor of the outer tunnel and to extracellular spaces and secretory and/or absorptive material which accu: mulates in these spaces.
Materials and Methods Mustache bats, Pteronotusparnelliiparnellii, from Jamaica, W.I., were used in this study. Animals were decapitated, the head cut in the midsaggittal plane and the cochleae rapidly removed. The cochleae were immersed in fixative consisting of 4% glutaraldehyde and 4% sucrose in 0.2 M s-collidine buffer, pH 7.3. The apex of each cochlea was opened and. the round window membrane was ruptured 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 four days. Both fixation and decalcification were carried out at room temperature, and the tissues prepared in this way were subsequently studied as surface preparations or further processed for LM, TEM or SEM studies, For nuclear staining the entire cochlea was carefully dissected to remove overlying bone and tissue was then stained with gallocyanin [13]. After staining. the different turns and the hook region were isolated and the specimens were dehydrated in an ethanol series, placed in xylene and then mounted on a slide with DPX and coverslipped. With the light microscope the different cellular elements of the organ of Corti could be easily recognized on the basis of their position and organization in rows. In order to obtain information on the population density and distribution of Boettcher’s cells, the images of the nuclei were projected onto paper with the aid of a drawing tube and a montage of the nuclei within the spiraling organ of Corti was
93
constructed; nuclear densities were counted per 0.1 mm length from the hook to the apex. Montages and counts were made from the different regions in six cochleae. Tissue to be sectioned for light microscopy was partially dehydrated in an ethanol series of 50% 70% and 95% infiltrated with glycol methacrylate (GMA) for lo-24 h and then embedded in fresh GMA [I]. 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 cochleae, cut in different planes, were processed in this way and the material was used to assess intercellular relationships and light microscopic features. For SEM observations of the cells of Boettcher the overlying Hensen’s cells were removed from the fixed material by treating the tissue with 1% trypsin for various periods of time [IO]. The tissue was then dehydrated with a graded series of acetone, critical point dried, coated with gold and examined with an ETEC scanning electron microscope. Tissue to be used for TEM was rinsed in 0.2 M s-collidine buffer, postfixed 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 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 cut, oriented and affixed 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 and lead citrate [22] and examined with a Joel 1OOB transmission electron microscope. The following descriptions of ultrastructural details are based on micrographs obtained from all turns of the cochleae.
Results The relationships of the cells of Boettcher to other cells in the organ of Corti of Pleronorus are shown schematically in Fig. 1. The cells are situated on the surface of the basilar membrane at or near the junction of the pars pectinata and the spiral ligament. They are bounded on the side toward the modiolus by a single row of cells which are morphologically distinct from all others in the organ of Corti; in accordance with their position these cells will be referred to as the basal cells of the outer tunnel or simply tunnel floor cells. On the side toward the spiral ligament are the cells of Claudius and above Boettcher’s cells are the cells of Hensen. These intercellular relationships are maintained throughout the cochlea. In this report only the relationships of Boettcher’s cells to each other, to the basilar membrane and to the basal cells of the outer tunnel will be considered. After trypsin digestion of the overlying tissue, the cells of Boettcher were observed in SEM micrographs as a continuous cord of cells’(Fig. 2A). Along the
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Pig. 1. Schematic diagram of cellular elements associated with the outer tunnel of the organ of Corti in Pfero!rofusp. putwellii. Illustrated are the relationships of Boettcher’s cells (B) to the pars pectinata of the basilar membrane (PP). the outer tunnel (OT). the basal cells of the outer tunnel (BOT). the cells of Deiters (D). the cells of Hensen (H). and the spiral ligament (SL). Also shown are several outer hair cells (OHC) and the cell which forms the roof (tectum) of the outer tunnel (TOT).
inside edge of the cord (Fig. 2A) were the nuclei of the tunnel floor cells and in places where these were removed (Fig. 2B) there were irregular accumulations of material interposed between the cells of Boettcher and the adjacent row of Deiters’ cells. From TEM and LM micrographs it was determined that the cells of Boettcher had spherical nuclei 4-5 pm in diameter. Although irregular in shape, the cells themselves measured approximately 6-8 pm in width and 6-7 pm in height (Fig. 3). TEM micrographs showed the same general characteristics that have been described for other species. This included large numbers of microtubules throughout the cytoplasm (Fig. 4); both bundles and individual tubules appeared to be randomly oriented but occasionahy a bundle was seen encircling the nucleus, as described by Kimura (161. The diameter of these tubules, whether in bundles or isolated was approximately 25 nm. The cytoplasm also contained mitochondria, lysosomes, a Golgi apparatus, vacuoles, vesiculated bodies and lipofuchsin granules; many free ribosomes were scattered throughout the cytoplasm. These ultrastructural features can be seen in several of the micrographs (Figs. 4-7) included in this report. In sharp contrast to the dense cytoplasm of Boettcher’s cells, the cytoplasm in all adjacent cells was almost completely devoid of any organelles (Fig. 3A). The cytoplasm (Figs. 3B and 5) and the spaces around the tunnel floor cells (Fig. 7) were crowded with clear, irregularly shaped vesicles and vacuoles of various sizes. Some of these appeared to be membrane bound, but the exact boundaries of the cells and vacuoles were difficult to assess. The cytoplasmic organelles of the tunnel floor cells included lysosomes and mitochondria and almost all of these were crowded into the basal portion of the cells. The nuclei were very irregular in shape and the shape seemed to. be influenced to a large degree by the pressure applied by surrounding vacuoles; however, deep indentations were also observed (Fig. 5). The average
Fig. 2. SEM micrographs of the ceils of Boettcher in the basal turn. These micrographs were obtained by disrupting and removing overlying tissue with trypsin. In A note the cord-like appearance of the cells of Boettcher (B) and their bulging nuclei (arrows); the nuclei of the adjacent tunnel floor cells are also evident (arrowheads). 1320 X In B some of the material which accumulates along the inner side of the cord of Boettcher’s cells can be seen next to the third row of Deiters’ cells (D). 3300X.
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Fig. 3. TEM micrographs of the cells of Boettcher. A shows a section transverse to the long axis of the cord. Note the relationship of three Boettcher’s cells (B) to each other and to the basilar membrane (BM) (beginning of the second turn). The arrow points to the thin osmophilic layer of material that underlies Boettcher’s cells and the tunnel floor cells. 6600X. B shows a longitudinal section along the cord of Boettcher’s cells in the apical turn. 5700X.
Fig. 4. Illustrations of the junctions between adjacent Boettcher’s cells. In A the types of junctions that occur extensively in the nuclear and supranuclear portions of each cell are evident. 19000X. B shows an example of the elaborate, microvillus-filled channels that occur near the bases of the cells. Note the dark material (M) which lies within the channel and the numerous microtubules within the cytoplasm of the cells (apical turn). 24000X.
Fig. 5. Microvillus-filled channels and their relationship to extracellular spaces near the tunnel floor cells. A shows a section parallel to the basilar membrane through the channels and illustrates the connections (arrow) of the channels with the extracellular spaces filled with darkly staining material. The clear spaces with vesicles represent the bases of the tunnel floor cells. 20000X. B shows a section at a slightly higher level in the cord. Note the deep cleft in the nucleus of the tunnel floor cell and the large clear vesicles (apical turn). 8000X.
Fig. 6. TEM micrographs of pore-like canals that exist between the microvillus-filled channels (C) and the basilar membrane (BM). A shows a longitudinal section through a canal containing the basilar membranelike material (basal turn). Note the many microtubules in this micrograph. 24650X. B is a transverse section through several channels and canals in the second turn. Continuity of the material with the upper layer of the basilar membrane can be seen. 23 250 X .
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diameter of the nuclei was about 7pm and thus they were significantly larger than the nuclei of Boettcher’s cells. Because of their size and distorted appearance the nuclei of the tunnel floor cells could be easily distinguished from those of the other cells when examined in whole mount preparations stained with the nuclear stain, gallocyanin. From such preparations, as well as serial sections, it was observed that both the tunnel floor cells and Boettcher’s cells occurred throughout the cochlea. The density of the tunnel floor cells was constant per unit length from the beginning of the hook to the apex, with about 10 cells per 100 pm. The density of Boettcher’s cells changed slightly. There were only two rows of cells in the hook region resulting in 20-30 cells per 100 pm; the number gradually increased to 35-40 cells per 100 pm and this was the number found throughout most of the remainder of the cochlea. There were, however, two regions with approximately 50 cells per 100 pm; these were from the middle to the distal end of the sparsely innervated region [8] and at the apex. The structure of the intercellular junctions between adjacent Boettcher’s cells varied in different levels of the cord. Junctions adjacent to the nucleus (Fig. 4A) and at supranuclear levels were narrow. The intercellular junctions below the level of the
Fig. 7. Transverse section through the extracellular space between a Boettcher’s cell (B) and the basal cell of the outer tunnel (BOT’) in the hook region. Note the elongate microvilli protruding into the vesicle-filled space. This section is near the point of continuity of a microvillus-filled channel and the extracellular space. 15 000 X
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nucleus had numerous interdigitating microvilli (Fig. 4B). Sections through the lower part of the cord of Boettcher’s cells showed that the spaces between interdigitating microvillus surfaces were actually channels between adjacent cells. The channels were continuous with extracellular spaces between Boettcher’s cells and the tunnel floor cells (Fig. 5A). These spaces were always filled with a dark (osmophilic) substance that had the same staining properties as the layer of the basilar membrane on which the cells rested (Fig. 5A). This layer may represent a thick basal lamina (see Fig. 3A). When sections were cut through the cord at slightly higher levels, the darkly staining material was observed to lie between adjacent tunnel floor cells; this material apparently corresponds to the irregularly shaped clumps of material observed in SEM micrographs (Fig. 2). Sections cut parallel and perpendicular to the surface of the basilar membrane showed that there were not only wide intercellular spaces through which the basilar membrane material projected, but also that there were pore-like canals. The continuity of these small canals with the larger microvillus-filled channels was evident when sections were cut both transverse and parallel to the longitudinal axis of the microvillus-filled channels (Fig. 6). At the point of confluence of the microvillus-filled channels with the extracellular spaces many microvilli were seen to project freely into the extracellular spaces. The micrograph in Fig. 7 shows some of these microvilli and vacuole-like profiles that were often encountered in the extracellular space, in the tunnel floor cell cytoplasm and to a limited extent in the microvillus-filled channels.
Discussion
The present study provides new information about the cells of Boettcher which relates to: (1) their distribution and density; (2) the presence of contiguous intercellular channels and large extracellular spaces; (3) the existence of several different types of secretory or absorptive material within the channels and spaces; (4) a consistent relationship of basilar membrane material to the channels; and (5) a consistent relationship to an unusual cell type which forms the floor of the outer tunnel. The concept of a distinct cell type forming the floor of the outer tunnel was first advanced by Henson and Henson [ 111. Although more details concerning these cells will be forthcoming, it is necessary here to note their close association with the cord of Boettcher’s cells and with the large extracellular spaces that exist between these two cell types. Both cells exist side by side throughout the cochlea and seem to be associated with the same types of extracellular material. It is not known if tunnel floor cells are present in mammals other than bats or if they, or Boettcher’s cells, exist in isolation. The distribution and density of Boettcher’s cells in Pteronotus is markedly different from that in other mammals. In other groups where Boettcher’s cells have been described they have been limited to the basal turn. In other bats which we have examined (Tuduridu, Mormoops, Rhinolophus) the distribution of Boettcher’s cells and tunnel floor cells was similar to that in Pteronorus and, therefore, may be a
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characteristic feature of Microchiroptera rather than a feature unique to Pteronotus. Although the cells extend throughout the cochlea in the bats thus far examined, the number of rows is considerably less than in the cat where there may be up to 17 rows and in the squirrel monkey which has up to 20 rows in certain regions [ 151. In our study of the population density of these cells we were interested in determining whether there were regions where the density changed and if so, could they be correlated with the unusual hearing capacities of Pteronorus, the innervation pattern and/or the marked volumetric differences known to occur in the Scala tympani and Scala vestibuli. The relatively constant density and distribution of Boettcher’s cells, however, argues against a direct correlation with any known variations in these features. Ishiyama et al. [15] have suggested that the function and distribution of Boettcher’s cells might be related to the efferent endings and the products they release into the fluid-filled spaces of Nuel. Such endings are numerous in the basal turn and decrease toward the apex. The data obtained from Pteronotus show nothing to dispute such an association; however the European horseshoe bat, Rhinolophus ferrumequinum, has no efferent fibers to the outer hair cells [4] yet the cells of Boettcher are distributed throughout the cochlea. The microvillus-filled channels that are described in this report were only recognizable as channels when sections were made along the axis of the channels (Figs. 4 and 5A) or when the channels were transected (Figs. 6 and 7). Other investigators [5,15,16] have noted numerous interdigitating microvilli between adjacent Boettcher’s cells and projections of small amounts of basilar membrane material, but the existence of microvillus-filled channels which communicate extensively with the underlying surface of the basilar membrane and with other extracellular spaces has not previously been described. Their presence in other mammals, however, is strongly suggested by published micrographs [5,15,16]. The intercellular spaces between the bases of adjacent Boettcher’s cells, the numerous small channels and the presence of intercellular vesicles strongly support the suggestion of Ishiyama et al. [15] that the cells have an important absorptive function; it would appear that whatever is absorbed must pass through the basilar membrane or be part of it. It is interesting to note that if the entire basilar membrane surface of Boettcher’s cells were covered with microvilli, then the cord of cells could not be firmly’anchored to the vibrating surface. In this case the biological solution seems to be the creation of the porous surface which permits the material to collect in the microvillus-filled channels. This arrangement appears to be unique to the organ of Corti. We do not know the nature of the material that may be secreted or absorbed by the cells of Boettcher, but there is a close association with a darkly staining (osmophilic) substance that is clearly a constituent of the deepest layer of the basilar membrane. The cells are also associated with clear, vesicle-like material. It may be that the cells of Boettcher are associated with the maintenance and turnover of the basilar membrane. Perhaps there is a constituent of the membrane that occurs only in specific parts of the cochlea in many mammals, and is important in governing the vibratory properties of the system.
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Acknowledgement This work was supported by USPHS Grant 12445. References 1 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, 71-97. 2 Boettcher, A. (1869): Ueber Entwicklung und Bau des Gehorlabyrinths nach Untersuchungen an Saugetieren. Novorum Actorum Academiae Caesareae Leopoldino-Carolinae Germanicae Naturae Curiosorum 35. I-203. 3 Brodmann, G. and Giebel, W. (1978): Histochemische Untersuchungen zur Funktion und Verteilung der Boettcherschen Zellen in der Goldhamstercochlea. Arch. Oto-Rhino-Laryngol. 220, IOS- I 16. 4 Bruns, V. and Schmieszek, E. (1980): Cochlear innervation in the greater horseshoe bat: Demonstration of an acoustic fovea. Hearing Res. 3.27-43. 5 Duvall, A.J. 111 and Sutherland, C.R. (1970): The ultrastructure of the extrasensory cells in the cochlear duct. In: Biochemical Mechanisms in Hearing and Deafness, pp. 149-170. Editor: M.M. Paparella. Charles C. Thomas, Springfield, 111. 6 Giebel. W. and Brodmann, G; (1977): Histochemie der Esterasen und Endopeptidasen im Innenohr. Arch. Oto-Rhino-Laryngol. 216, 521-522. 7 Grinnell. A.D. (1967): Mechanisms of overcoming interference in echolocating animals. In: Les Systems Sonars Animaux Biologie et Bionique Tome I, pp. 451-480. Editor: R.G. Busnel. Laboratoire de Physiologic Acoustique INRA-CNRZ, Jouy-en-Josas 78, France. 8 Henson. M.M. (1973): Unusual nerve-fiber distribution in the cochlea of the bat, Pteronorur p. purnellii (Gray). J. Acoust. Sot. Am. 53. 1739-1740. 9 Henson, M.M. (1978): The basilar membrane of the bat, Preronotm p. purnellii. Am. J. Anat. 153, 143-157. 10 Henson. M.M. and Henson, O.W. Jr. (1979): Some aspects of structural organization in the cochlea of the bat. Pferonorus p. purnellii. Scanning Electron Microsc. 1979/111. 975-982. I I Henson. M.M. and Henson, O.W. Jr. (1979): The supportive elements of the organ of Corti in the bat, Pteronorus p. purneilii. Presented at the Meeting of the Southern Society of Anatomists, Washington, D.C. Anat. Rec. (in press). 12 Henson, M.M. and Jenkins. D.B. (1981): The Scala tympani and the ultrastructure of the tympanic covering layer in the bat. Pteronotus porneliii pumellii. Abstracts of the Fourth Midwinter Research Meeting. Association for Research in Otolaryngology. p. 33. I3 Humason. G.L. (I 972): Animal Tissue Techniques. W.H. Freeman and Company, San Francisco. I4 Ishii. T. and Balogh, K. Jr. (1966): Acid phosphatase activity in the inner ear. Acta Oto-laryngol. 62, 185-192. 15 Ishiyama, E., Cutt, R.A. and Keels, E.W. (1970): Distribution and ultrastructure of the Boettcher’s cells in mammals. Ann. Otol. RhinoI. Laryngol. 79, 54-69. I6 Kimura. R.S. (1975): The ultrastructure of the organ of Corti. Int. Rev. Cytol. 42, 173-222. I7 Pollak. G.D.. Henson, O.W. Jr. and Johnson, R. (1979): Multiple specializations in the peripheral auditory system of the CF-FM bat, Pteronorus purnellii. J. Comp. Physiol. 131. 255-266. I8 Pollak. G.D., Henson. O.W. Jr. and Novick, A. (I 972): Cochlear microphonic audiograms in the ‘pure tone’ bat. Chilotaycteris purnellii purnellii. Science 176. 66-68. I9 Pye, A. (1966):The structure of the cochlea in Chiroptera. I. Microchiroptera: Emballonuroidea and Rhinolophoidea. J. Morphol. I 18. 495-S IO. 20 Suga, N. and Jen, P.H.S. (I 977): Further studies on the peripheral auditory system of ‘CM-FM’ bats specialized for fine frequency analysis of Doppler-shifted echoes. J. Exp. Biol. 69, 207-232. 21 Suga, N.. Simmons, J.A. and Jen, P.H.S. (1975): Peripheral specialization for fine analysis of Doppler-shifted echoes in ‘CF-FM’ bat, Preronorus pumellii. J. Exp. Biol. 63. I6 I - 192. 22 Venable. J.H. and Coggeshall. R. (1965):A simplified lead citrate stain for use in electron microscopy. J. Cell Biol. 25. 407-408.