Ultrastructural differentiation of the first hensen cell in the gerbil cochlea as a distinct cell type.код для вставкиСкачать
THE ANATOMICAL RECORD 240:149-156 (1994) Ultrastructural Differentiation of the First Hensen Cell in the Gerbil Cochlea as a Distinct Cell Type SAMUEL S. SPICER AND BRADLEY A. SCHULTE Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, South Carolina ABSTRACT Background: The mammalian cochlea contains beneath and lateral to outer hair cells, several types of supporting cells. The function of these cells has not been explained beyond providing a structural base. Methods: The supporting cells of gerbil cochlea were examined by electron microscopy with a view to elucidating their biologic activity on the basis of cytologic structure. Results: Ultrastructural examination differentiated the laterally located Hensen cells from their medial neighbor connected to the third Deiters cell. The latter cell formed a cover to the outer tunnel between Hensen and Deiters cells, appeared not to reach the basilar membrane, and exhibited a denser cytosol and more mitochondria, compared to Hensen cells. In these respects the cell observed here to cover the outer tunnel, corresponded with the tectal cell described by Henson et al. (1983) in the mustache bat, but not heretofore documented in other animals. Conclusions: This distinctive cell in the gerbil differed in displaying unique villus-like structures which projected from the basomedial surface and are referred to as fimbriae. The fimbriae and interspersed filopodia largely filled outer tunnel space and expanded the cell’s basal surface. The amplification of basal plasmalemma by fimbriae and their content of mitochondria testify to a role for the tectal cell in ion resorption and an influence on ion content and volume of outer tunnel fluid. Q 1994 Wiley-Liss, Inc. Key words: Cochlea, Supporting cells, Morphology, Ion transport, Ultrastructure, Gerbil, Outer tunnel The fine structure of the lateral supporting cells in the mammalian cochlea, including Deiters and Hensen cells, has been investigated in several studies. Deiters cells support outer hair cells on a cup-shaped apical surface and project a phalangeal process apically into the reticular lamina (Smith and Dempsey, 1957; Engstrom and Wersall, 1958; Iurato, 1961: Kimura, 1975, 1984; Smith, 1978). Dieters cells contain numerous organelles including mitochondria and the rosette complex in their apical compartment (Spicer and Schulte, 1993), and in their basal portion a microtubule stalk (Angelborg and Engstrom, 1972) which apparently contributes to the mechanical support of overlying hair cells. Hensen cells, located lateral to Deiters cells and the outer tunnel of Corti, consist of lucent flocculent cytosol enclosing sparse organelles-mainly mitochondria and osmiophilic bodies (Engstrom and Wersall, 1958; Kimura, 1975, 1984; Santi, 1988). Hensen cells have been described as showing club-like or polypoid projections toward the outer tunnel and becoming thin in the region connecting with the phalangeal process of the third Deiters cell (Kimura, 1975). Prominent microvilli (c) 1994 WILEY-LISS, INC which have been interpreted as indicating a resorptive function, characterize the apical surface of Hensen cells (Engstrom and Wersall, 1958; Kimura, 1984). Consisting of few organelles and mainly a translucent cytosol which can be presumed to be highly hydrated, Hensen cells are difficult to preserve. Presumably, for this reason, there are few published illustrations of the fine structure of these cells. The present study aimed a t enquiring further into the structure of the lateral supporting cells in the gerbil cochlea. Evidence was obtained that the cell in the area assigned to the first Hensen cell differs in structure and presumably in function from the more lateral Hensen cells and comprises a distinct cell type. This cell in gerbil cochlea resembles in some but not other structural features, the cell observed in the mustache Received February 7, 1994; accepted March 23, 1994. Address reprint requests to Dr. Samuel S. Spicer, Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. 150 S.S.SPICER AND R.A. SCHULTE Fig. 1. Toluidine blue stained thick section showing a cell (TC) covering the outer tunnel (OT) and presumed to correspond with the tectal cell of the bat. Segments and processes of the cell fill much of the tunnel. Cytosol in the first Hensen cell (HC-1)is lighter than that in the third Deiters cells (DC-3) it borders closely. Hensen cells lie below and lateral to the outer tunnel and rest on basilar membrane (BM). Laterally, the Claudius cells (arrows) overlie Boettcher cells (BC), basilar membrane, and outer sulcus cells (OSC).4 kHz. x 900. bat by Henson et al. (1983) and designated the tectal cell. window. The cochleas were dissected free and left overnight with fixative, in the refrigerator. The scalae were subsequently flushed gently with phosphate buffer and perfused with 1% osmium tetroxide in phosphate buffer. This postfixation was terminated after 30 min by flushing the scalae with distilled water. Following fixation, the inner ears were trimmed of excess bone and decalcified by perfusion with and then immersion in 0.12M EDTA at pH 8.0. EDTA solutions were changed daily for about three days until decalcification was complete. Specimens were then dehydrated by perfusion and immersion with graded alcohols and propylene oxide, and embedded in Epon LX-112 resin. Before the resin had completely hardened, the cochleas were bisected lengthwise and cut into half turns which were re-embedded in Epon. Radial slices were cut with a razor blade from the cochlear regions encoding 0.5 to 10 kHz (Tarnowski e t al., 1991). These slices were re-embedded in Epon. Thick sections and adjacent thin sections were taken a t several levels, 25 pm apart from each epoxy block, and the thick sections were stained with toluidine blue. Ultrathin sections were selected for electron microscopic examination and stained with uranyl acetate and lead citrate MATERIALS AND METHODS The six Mongolian gerbils (Meriones unguiculatus) examined here were housed in a low-noise room from birth to sacrifice a t 3-6 months of age. Similarly aged animals from the gerbil colony maintained in this facility have consistently shown normal hearing as judged by evoked potentials of brain stem and auditory nerve (Schmiedt, 1989; Mills et al., 1990). Procedures for the care and use of animals were approved by the Animal Use Committee of the Medical University of South Carolina under NIH Grant DC 00422. The gerbils under urethane anesthesia were perfused with minimal pressure via a cardiac catheter employing 10 ml of room temperature normal saline that contained 0.1% NaNO,. After exsanguination, they were perfused with 50 ml of room temperature fixative fluid containing 4.0% paraformaldehyde and 2.0% glutaraldehyde in 0.1M phosphate buffer, pH 7.4. The bullae were then opened, the stapes was removed, the round window was perforated, and 1.0 ml of the fixative was injected gently into the scalae via the round COCHLEAR TUNNEL CELLS 151 Fig. 2. The tectal cell (TC) forms a roof over the outer tunnel and attenuates toward connection a t a tight junction with the Deiters cell phalanx (P)a t the lateral limit of the reticular lamina. Filopodia (F) and fimbriae (arrows) extend from the cell into the tunnel. The fimbriae reach the base of the phalanx and lateral surface of the third Deiters cell. A rosette complex tRC1 occupies apical cytosol near the Deiters cell’s contact with fimbriae. 4 kHz. x 5,000. Flg. 3. In a microscopic field below that of Fig. 2, minute processes presumably derived from the tectal cell (TC) fill much of the outer tunnel. These fimbriae reach the lateral surface of the third Deiters cell IDC) but not the surface of Hensen cells tHC) below. 4 kHz. x 2,500. when the adjacent thick section showed a longitudinal view of well-preserved organ of Corti. Sections were viewed ultrastructurally from six animals a t one or more mid-frequency regions. the apicolateral corner of the outer tunnel in the position generally assigned to the first Hensen cell (Figs. 1, 2, and 4).This cell differed, however, from the Hensen cells lateral to it in both location and cytologic structure, and resembled more closely the tectal cell of the bat (Henson et al., 1983).It joined a Hensen cell laterally a t an apical tight junction and sent a tapering process medially to a tight junction with the phalanx of RESULTS An irregular cell profile often enclosing a roughly spherical nucleus with dispersed chromatin, occupied 152 S.S. SPICER AND B.A. SCHULTE Fig. 4. A segment ( S ) of a cell covering the outer tunnel (OT) contains an apical nucleus and reaches upward toward the reticular lamina out of the field. The tunnel’s cover cells (TC) possess fairly numerous, widely distributed mitochondria and project filopodia, and numerous fimbriae into the tunnel toward the apicolateral surface of the lateralmost Deiters cell. Rosette complexes (RC)lie beneath these surfaces. Fimbriae contain minute, dense mitochondria (arrows). 500 kHz. x 4,250. COCHLEAR T U N N E L CELLS 153 Fig. 5. Tectal cells (TC) a t the top contain relatively dense cytosol and extend fimbriae into the outer tunnel (OT) in a tangential section of the organ of Corti. Deiters cells (DC) under outer hair cells (OHC) contain the rosette complex (RC) and many mitochondria in their upper compartment and granular cytosol, and a microtubule stalk (S) in the bottom part. The first Hensen cell (HCI) and more lateral Hensen cells display lucent, flocculent cytosol, and infrequent mitochondria and adjoin neighbors closely on a fairly regular boundary. Amorphous material separates upper and lower fibrous bands in the basilar membrane (BM). 4 kHz. x 2,250. Fig. 6. Fimbriae of a tunnel cover cell enclose lucent cytosol and are bordered closely by round, dense profiles of presumed microvillar projections from the fimbrial surface (arrows).10 kHz. x 12,500. the third Deiters cell, thus delimiting the outer tunnel space apically (Figs. 1 and 2). The lateral surface of segments of the cell covering the outer tunnel in the gerbil fitted against Hensen cells (Figs. 1 and 5). Profiles of these cells were not observed in continuity from a n apical front on endolymph, to a contact with the basolateral surface of the third Deiters cell or with basilar membrane. Instead, sections showing the presumed tectal cell at the roof of the tunnel also disclosed a segment of a cell with structural features of a Hensen cell adjoining the third Deiters cell along its basolat- era1 plasmalemma and resting on the basilar membrane (Fig. 5 ) . The cell covering the outer tunnel in the gerbil possessed denser cytosol than the more laterally situated Hensen cells, and disclosed, moreover, more numerous mitochondria which were distributed throughout the cytoplasm rather than a t the margins (Figs. 4 and 5). The most distinctive feature of the gerbil’s tunnel cover cell, however, concerned its highly expanded basal and medial surface (Figs. 1-91. The cell extended numerous villus-like projections, referred to as fim- Flg. 7. Elongated profiles of longitudinally sectioned fimbriae protrude into the outer tunnel (OT) toward the third Deiters cell (DC). TC, tectal cells. x 2,250. 154 S.S.SPICER AND R.A. SCHULTE Fig. 8. Fimbriae infiltrate tunnel space between a tectal cell and Deiters cell. The filamentous meshwork (arrow) of a rosette complex together with marginated mitochondria, lie beneath the Deiters cell plasmalemma contacted by fimbriae. 4 kHz. x 7,500. Fig. 9. Profiles of fimbriae enclose membrane limited bodies, possibly lysosomes (arrowheads),and minute mitochondria. The mitochondria reveal cristae separated by sparse matrix. Microvillar extensions protrude from some fimbriae (arrows). 1 kHz. x 33,750. briae, along with irregular filopodia, into the outer tunnel. The fimbriae and filopodia filled much of the tunnel space reaching the apicolateral surface of the third Deiters cell, often over a n underlying rosette complex (Figs. 2, 4,and 8). Minute cell processes presumed to be microvillar extensions from the surface, bordered some fimbriae (Fig. 6). The diameter of the fimbriae ranged mostly between 0.2 and 0.5 pm but occasionally reached up to 1.0 pm. The fimbriae enclosed lucent cytosol, sparse membrane limited bodies, and dense, exceptionally small mitochondria (Figs. 6 and 9) which measured about 0.2 pm in diameter. A full profile of the most medial (first) Hensen cell was rarely observed, presumably because of a n arched contour and orientation a t a n angle to that of Deiters cells. The few full images obtained showed that the presumed first Hensen cell maintained extensive basomedial contact with the basolateral plasmalemma of the third Deiters cell, basal contact through basement membrane with basilar membrane, and apicomedial contact with segments of the tectal cell or outer tunnel space, while also fronting apically on the scale media. More often, apical segments of Hensen cells were observed bordering tectal cells, and separate basal seg- ments (Fig. 5) were encountered in approximation to Deiters cells and basilar membrane. Although not in continuity, these profiles appeared similar cytologically, and were assumed to represent upper and lower regions of the first Hensen cell. The first Hensen cell thus formed the basal limit and part of the lateral boundary of the outer tunnel. The somewhat irregular lateral plasmalemma of the second Hensen cell approximated its neighbors’ surface closely except a t occasional focal separations. DISCUSSION Several structural features afford evidence for differentiating the cell in the first Hensen cell area of gerbil cochlea a s a separate cell type. The body of this cell joined laterally to a Hensen cell and its thin medial process reached the reticular lamina forming a cover to the outer tunnel a s shown schematically in Figure 10. The cell also displayed denser cytosol and more numerous and widely distributed mitochondria compared with Hensen cells. In these respects, the cell covering the outer tunnel in gerbil cochlea resembled that observed by scanning and transmission electron micros- 155 Cells Deiters Cells Fig. 10. Diagramatic representation of cells delimiting lateral tunnel spaces in the organ of Corti. copy by Henson et al. (1983) in the mustache bat, Pteronotus p . parnellii, and referred to as the tectal cell. Tectal cells have apparently not been demonstrated or referred to in a n animal other than the bat. No single profile of this distinctive cell yet observed has revealed a surface exposed to the scala media, contact with the basolateral plasmalemma of the third Deiters cell, and apposition to the basilar membrane. The cell in the gerbil like that in the bat can therefore be presumed not to reach basilar membrane a s do Hensen cells. However, Hensen cells were only rarely observed in full length profile from scala media to basilar membrane because of curvature and orientation. Nevertheless, it appears doubtful that a cell profile facing scala media and interpreted as a tectal cell actually possessed continuity out of the plane of section with a basal cell profile bordering a Deiters cell and the basilar membrane and interpreted as a Hensen cell. The reason for this view lies in the invariably greater cytosolic density and abundance of organelles in profiles of the tunnel covering cell, compared with the presumed Hensen cell segment at the base. Conversely, a question arose whether a partial cell profile bordering the base of the third Deiters cell and resting on basilar membrane, and a separate profile bordering the tectal cell and fronting on scala media, represented basal and apical segments of Hensen cells connected a t a level outside that of the plane of section. Ultrastructural findings of Henson et al. (1982) suggested that the basally located cell profiles in the bat constitute a separate cell-type termed the tunnel floor cell. Present observations in the gerbil favor interpreting the cell contacting the third Deiters cell and basilar membrane a s part of a Hensen cell that reaches the scala media a t another level. This interpretation rests on the content of similar lucent, flocculent cytosol and sparsity of organelles other than a few mitochondria in both the lower and the upper segments, and the infrequent observation of a first Hensen cell extending from basilar membrane to scala media. The fimbriae decorating the basomedial surface were the most definitive structures characterizing the cells covering the outer tunnel. In this respect, the cover cell a t the roof of the tunnel in the gerbil cochlea differed from the bat’s tectal cell (Henson e t al., 1983) which lacked such plasmalemmal amplification. To our knowledge, this specialized surface adaptation has not been encountered on any other cell. Such a fimbriated cell appears not to have been described heretofore, in cochlea, possibly because of differences between gerbils and the other genera examined. Expansion of the plasma membrane inward or outward from the surface supplies increased area for flux of fluid and solute to or from the cell. Surface expanded by infoldings of apical or basal plasmalemma, a s for example in gastric parietal cells or renal and salivary gland ductal epithelium, commonly utilizes a n ATPase pump to increase efflux of a specific ion against a gradient. However, the cells a t the roof of the outer tunnel lacked a cytochemically detectable level of Na,KATPase (Schulte and Adams, 1989) or H,K-ATPase (S.S. Spicer, A.J. Smolka, and B.A. Schulte, unpublished data) and thus failed to yield evidence of such pump activity. On the other hand, amplified plasmalemma in the form of microvilli protruding from the cell can facilitate a less energy dependent resorption down a gradient. The extensive evaginations of the fimbriated cell’s plasmalemma into the lumen of the outer tunnel points to a probable similar function a t this site. The fimbriae differ from microvilli of brush border cells, however, in their larger dimensions and content of abundant cytosol and exceptionally minute mitochondria. The mitochondria in the fimbriae testify to a transport process with an ATP requirement intermediate between that in striations of renal and glandular duct cells and that in microvilli of intestinal or renal brush borders. The fimbriated surface of cover cells provides evidence of resorption by these cells, implying they affect ion content and volume of fluid in the outer tunnel. 156 S.S. SPICER AND B.A. SCHULTE The fimbriae can be viewed in this light as resorbing an ion, possibly K + from outer tunnel fluid for transport to Hensen cells and ultimately Claudius cells through the gap junctions that have been demonstrated between inner ear supporting cells (Iurato e t al., 1976; Nadol, 1978; Santos-Sacchi, 1987). This K' uptake could counter balance the sound induced efflux of K' from outer hair cells and nerves into the outer tunnel (Johnstone et al., 1989) and impede K' leakage from endolymph into the tectal cells and Hensen cells. Largely lacking apical microvilli, the fimbriated cells are presumably not concerned, as are neighboring Hensen cells, with resorption from endolymph, and for this reason and their apparent failure to reach basilar membrane appear not to contribute directly to fluid or ion translocation between endolymph and perilymph. ACKNOWLEDGMENTS The authors appreciate the valuable technical and secretarial assistance of Mrs. Nancy Smythe and Mrs. Leslie Harrelson. This work was supported by NIH Grants DC 00422 and DC 00713. LITERATURE CITED Angelborg, C., and H. Engstrom 1972 Supporting elements in the organ of Corti. Acta Otolaryng. Suppl., 301.49-60. Duvall, A.J., and C.R. Sutherland 1970 The ultrastructure of the extrasensory cells in the cochlear duct. 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