- Email: [email protected]
The cell coat of inner ear sensory and supporting cells as demonstrated by ruthenium red
281
~fecrrrng Rewarc~h, 17 (1985) 281-288
Elsev1er
HRR 00589
The cell coat of inner ear sensory and supporting ruthenium red * Norma
Slepecky
(Received
’
cells as demonstrated
and Steven C. Chamberlain
21 December
1984: accepted
2X February
by
’
1985)
The ultrastructure of the cell coat of sensory and supporting cells of chinchilla and lizard inner ears was studied using ruthenium red. On the apical surface of both cell types, in both animals, the glycoproteins in the cell coat stain posltwely wth this cationic dye. The apical surface of the sensory hair cells displays no regional variations in cell coat thickness. The uniform stainmg along the length of the stereocilia is not influenced by the normal presence or absence of a tectorial membrane. Although no micro-domains in the glycoproteins that stam positively wth ruthenium red were observed that might correlate with the ultrastructural localization of sites of initiation of the transduction event. the cell coat material on the apical cell surface might play an important role in sequestering ions (particularly calcium) which are required for the transduction process. cochlea.
cell coat, cochlear
hair cell, ruthenium
red
Introduction The surface of almost all cells is covered with a fuzzy layer of mucopolysaccharide, glycoprotein and glycolipid material, associated with and forming a part of, the plasma membrane [3]. Histochemical studies of this layer [18,4] have suggested that the components of the cell coat may be responsible for the way the cell reacts to its external environment. The cell coat may isolate the cell from its surrounding medium and create in effect, a microenvironment. The presence of acidic glycoproteins and glycolipids contributes to a net negative cell surface charge which plays a role in cell-cell and cell-substrate interactions. By virtue of its negative charge, this layer can also sequester cations immediately adjacent to the cell membrane [17]. By limiting or promoting availability of these ions, the cell coat can regulate cell membrane conductance. * All work described in this report was carried out in full compliance with the National Institutes of Health Guidelines for the Use of Experimental Animals. 037%5955/85/$03.30
0 1985 Elsevier Science Publishers
The presence of cell coat material has previously been noted on inner ear cells. The stereocilia of the outer hair cells stain positively for periodate-reactive mucopolysaccharides with the periodic acid Schiff’s (PAS) reaction [l]. Observations with the electron microscope show fibrils surrounding and between adjacent stereocilia [8,2,24]. Cationic probes such as ruthenium red [29] and cationic ferritin [9] stain a prominent cell coat composed of acidic glycoproteins. These findings have led to two suggestions about the function of the surface components of the stereocilia. First, by cross-linking adjacent stereocilia, the cell coat serves to hold the stereocilia together in a bundle so when a stereocilia bundle is deflected, all stereocilia within that bundle move as a group. Second, because of the electronegativity of its acidic groups, the cell coat serves to maintain, by repulsion, a distance between adjacent stereocilia, preventing close contact and preventing the cell membranes from fusing. Recent evidence suggests that stereocilia play an important role in the iransduction process whereby a mechanical stimulus causes depolariza-
B.V. (Biomedical
Division)
282
tion of the hair cell and the generation of a receptor potential. Deflection of the stereocilia initiates this event [15] and at some place along the apical surface of the sensory cell, ion channels specific for positive ions are opened and current flows into the cell [6]. The actual site for this change in membrane conductance has yet to be determined. Initial suggestions based on stereocilia and cuticular plate anatomy included: a deformation-sensitive membrane on the apical cell surface adjacent to the point of insertion of the tallest stereocilia and lying above the basal body and the cuticle-free region of the cuticular plate [ll]; the tapered region of each stereocilium where it pivots when deflected [9]; the lateral surface of the stereocilia where a larger area of ion-permeable membrane would be exposed when stereocilia separate during deflection [19]; and distortion of the cross-links which connect the tips of one row of stereocilia to the adjacent, taller stereocilia in the next row [24]. Recent evidence suggests that the site for changes in the ion conductance mechanism is located at the tips of the stereocilia [12], that all the stereocilia are involved, and that there may be only a few transduction channels per hair cell [13,23,25]. Attempts have been made to localize the site of transduction ultrastructurally, based on the assumptions that such ionic channels might be represented by intramembranous proteins and that their activation requires calcium. Observation and analysis of freeze-fractured stereocilia membranes, however, shows only a random distribution of intramembranous particles. There appear to be no regional specializations along the apical or stereocilia membrane that correspond to proteins that might function as ion channels during transduction [lo]. Attempts to use oxalate or antimonate precipitates to mark sites of calcium-binding proteins have given positive results but the distribution of electron reaction product at specific sites along the apical cell surface is not constant across species [22]. Since the cell coat may also play a role in determining ion conductance, it is of interest to study the cell surface along the apical surface of inner ear cells. Micro-domains of specialized membrane function might be represented by variations of thickness in the cell coat material. For this reason, we have used ruthenium red to char-
acterize the cell coat material and to mark Its distribution along the apical surface of the organ of Corti. In order to preclude any influence of the mucopolysaccharides from the tectorial membrane [7,30] in the study of the cell coat material on the inner and outer hair cell stereocilia, we have also studied the sensory cells from the inner ear of the side-blotched lizard Uta .stansburiana where the cells having stereocilia associated with a tectorial membrane are in a separate region of the basilar papilla from those without a tectorial membrane [211. Materials and methods Inner ears from four chinchillas and four sideblotched lizards (Uta stansburiana) were processed for demonstration of the cell coat using ruthenium red. Those from the same number of animals were processed as controls. Chinchillas were decapitated, the temporal bones removed from the skull and openings made at the apex, round window, and oval window. Care was taken to make sure that Reissner’s membrane was punctured in both the apical and basal regions. Lizards were decapitated, the temporal bones exposed, and openings made in the round and oval windows and in the ampullae of the semicircular canals. Initially, a solution containing 2.5% glutaraldehyde, 0.1 mM MgCl, and 0.125 M phosphate buffer at pH 7.2 was perfused through all openings. Perfusion was continued for 5 min. After this time, temporal bones were placed in scintillation vials containing 10 ml of each subsequent solution, and the vials were placed on a rotator to ensure continuous agitation. Temporal bones were washed in 0.1 mM MgCl, in 0.125 M phosphate buffer at pH 7.2 f 500 pg/ml ruthenium red for 1 h. Then they were postfixed with 2% 0~0, t_ 500 pg/ml ruthenium red in 0.1 mM MgCl, in 0.125 M phosphate buffer at pH 7.2 for 2 h in darkness. Temporal bones were again washed in buffer + ruthenium red for 1 h, then dehydrated through ethanol to propylene oxide and embedded in Araldite. Cochleas were dissected, quarter turns reembedded and trimmed, and thin sections cut and examined unstained with the transmission electron microscope.
283
Results In both the chinchilla and lizard inner ear, sensory and supporting cells display a cell coat which stains positively with ruthenium red. In the chinchilla cochlea, the inner and outer sensory hair cells are located close together on the basilar membrane in the organ of Corti. The stereocilia of both
la
types of hair cells are adjacent to, if not embedded in, the tectorial membrane. In unstained sections of cochleas treated with ruthenium red, it can be seen that the apical surface of the sensory and supporting cells have a uniform layer of electron dense material external to the cell membrane. Outer hair cell stereocilia bundles, cut in crosssection, display this ruthenium red positive
lc
lb Fig. 1. Cross-sections through outer hair cells of the chinchilla cochlea. (la. lb) Electron micrographs of unstained sections of stereo&ha from cochleas processed with ruthenium red. Cell coat material covers the surface of each stereocilium and large accumulations of material connect adjacent stereocilia. (lc) Stereocilia from cochlea processed without ruthenium red. Cell coat material is not obvious and accumulations between adjacent stereocilia are not prominent. Magnifications: la, 24000 x ; lb. 48000 x : lc. 24000x.
284
material around the periphery of each stereocilium (Fig. la, b). In sections which are cut through stereocilia bundles at different points along their length, the coat appears at all levels. Near the tips there are larger accumulations between and connecting adjacent stereocilia. This material can also be observed connecting adjacent stereocilia in cochleas not treated with ruthenium red (Fig. lc), but the material stains prominently when ruthenium red is included in the processing solutions (Fig. 1b). Inner hair cell stereocilia cut in cross section also display a uniform layer of ruthenium red positive material which connects closely apposed stereocilia (Fig. 2a, b). When ruthenium red is omitted from the procedure, the fibrils are not stained and the inner cell stereocilia are separated by a space (Fig. 2~).
When the hair cells are cut longitudinally. the staining pattern along the apical surface of the cell and along the length of the individual stereocilia can be observed (Fig. 3a, c). Stereocilia from both inner and outer hair cells show a thin layer of ruthenium red positive material. There appears to be no variation in the thickness of the cell surface material at the apical surface of the cell, along the lateral membrane of the stereocilium, or at the stereocilium tip (Fig. 3b, d). The cross-bridge linking the tip of the shorter stereocilium with the longer one behind it (Fig. 3d) has been preserved and does not stain prominently with ruthenium red. The staining pattern is similar for the sensory and supporting cells of the basilar papilla of the side-blotched lizard. Two types of hair cells occupy different positions along the papilla. In the
Fig. 2. Cross-sections through inner hair cells of the chinchilla cochlea. (2a, 2b) Electron micrographs of unstained sections of stereocilia from cochleas processed with ruthenium red. Cell coat material covers the surface of each stereocilium and fib&s connect closely apposed stereo&ha - see also Fig. 3a. (2~) Stereocilia from cochlea processed without ruthenium red. Cell coat material is not obvious and stereo&ha appear separate. Magnifications: Za, 12CtOOX ; 2b, 48000 X : SC. 12000 X
2x5
center of the papilla is a region of two to three irregular rows of unidirectionally oriented hair cells with short stereocilia that are covered by a tectorial plate (Fig. 4a). At either end are two rows of bidirectionally oriented hair cells with long stereocilia that lack any association with a tectorial membrane (Fig. 4~). The sensory and supporting cells of the side-blotched lizard basilar papilla also display a cell coat which stains positively with ruthenium red. Again the stereocilia from both
types of hair cells display a uniform material along their lengths (Fig. 4b, d).
layer
of
Discussion The stereocilia of sensory hair cells have been implicated in the transduction process and it is of interest to know where along the apical surface of the sensory hair cells the actual transduction event occurs. It is known that a cell coat is present on
3b
Fig. 3.
Electron
Ruthenium
micrographs
of Ion~tud~naI
red positive material
there is no regional specialization Magnifications:
sections through
forms a uniformly
stereocilia
thick electron-dense
at the tips of stereocilia.
3a. 10200 X ; 3b. 30000 x ; 3c, 10200 x
:
(3a, 3b) Inner
3d, 30000 x
from chinchilla
cochlea
processed with ruthenium
layer on the apical surface of supporting hair cell stereocilia.
(3~. 3d) Outer
red.
and sensory cells:
hair cell stereocilia.
286
the apical surface of inner ear cells. Since the apical surface with its stereo&ha is the region that is thought to undergo ion conductance changes during transduction and since the cell coat is thought to be capable of regulating membrane conductance, further study of the cell coat seems
to be a logical approach to the study of sensory cell function. Histochemical probes such as ruthenium red have been used to characterize the cell coat components of various tissues and specific cell types. Acidic glycoproteins and glycolipids (with carbo-
Fig. 4. Bectron micrographs of sections through the basilar papilla of the side-blotched lizard Uta stamburiunu processed with ruthenium red. (4a) Region of hair cells with short stereocilia and tectorial membrane. (4b) Tips of short stereo&a show no speciakation of ruthenium red positive material. (4c) Region of hair cells with long stereocilia and no tectorial membrane. (4d) Tips of long stereocilia show no regional specialization of ruthenium red positive materiaf. Magnifications: 4a, 800 X : 4b, 30000 X : 4~. soox; 4d, 3oooox.
287
xyl and sulfate groups projecting out from the external surface of the plasma membrane) contribute to a net negative surface charge and it is these groups which are responsible for a ruthenium red positive reaction. Using this cationic dye, it has been determined that the thickness of the cell coat varies in different cells and tissues [18,4]. Within the olfactory system [20] sensory cells of the olfactory epithelium proper stain differently from those of the vomeronasal epithelium, and this difference may be reflected in the differences in odor perception by the two types of receptor organs. In addition, the cell coat of the sensory cells of the vomeronasal epithelium stains differently from that on the adjacent supporting cells. Individual cells may also display regions where thickness of cell coat varies. Capillary endothelial cells have such regions [18] and it is these micro-domains that are thought to correlate with regions of selective permeability. Previous investigators have noted the fibrillar nature of the extracellular material associated with the cells of the inner ear [8,29,7,2] and some have studied its nature and function using histochemical and anionic probes [1,29,9]. In this study we have characterized the cell coat material on inner ear cells using ruthenium red. Both supporting and sensory cells display a cell coat on their external surface. This coat appears of equal thickness on both cell types and also on inner and outer hair cell apical surfaces and stereocilia. Near the tips of the outer hair cell stereocilia large accumulations of ruthenium red positive-staining material are found connecting adjacent stereocilia within a bundle. When cochleas are processed with ruthenium red in the solutions, stereocilia of inner hair cells are connected by fibrils and are seen in close apposition, similar to their configuration when cochleas are processed for scanning electron microscopy. They appear separate in cochleas processed in the absence of ruthenium red (Fig. 2c; [16]). Since stereocilia are known to taper near their point of insertion into the cuticular plate, this observation must be treated with caution and the interpretation depends on the plane of section. In the specimens examined, inner hair cell stereocilia had been cross-sectioned close to the tips of the longest stereocilia, and not close to the region of
the taper. The apposition was also observed when stereocilia were sectioned longitudinally as shown in Fig. 3a and b. It is not yet known if this is because ruthenium red preserves the interactions at the tips of adjacent inner hair cell stereocilia, or if by neutralizing the electronegativity on the surface, it allows the stereocilia to move closer together than is normal. In either case, it may be another indication that the stereocilia of inner hair cells are different from the stereocilia of outer hair cells [28]. The stereocilia of both chinchilla and lizard show no differences in cell coat staining along their length which might indicate a region specialized for the initiation of the transduction event. Although others have had similar results when analyzing intramembranous particles [lo] and localizing calcium binding proteins [22], this does not prove that micro-domains in the cell surface do not exist. Pickles et al. [24] have preserved some of the cross-linking material that links the tips of the shorter stereocilia with the longer stereocilia behind them, by using glutaraldehyde without osmium postfixation, and have suggested that the stretching of these cross-bridges might deform the stereocilia membrane and initiate the transduction event. This material does not appear to be stained prominently with ruthenium red, so its components may be other than acidic glycoproteins. Other histochemical probes may prove useful in characterizing and localizing this material. As the ruthenium red-positive material appears to be distributed along the apical surface of the sensory and supporting cells regardless of their position with respect to the tectorial membrane, the cell surface may still play a role in transduction. Since acidic glyco-conjugates along the cell surface can sequester cations, it might be expected that the cell coat along the apical surface of these cells might create a cation-rich micro-environment [17] - particularly for calcium - an ion which is necessary for transduction [27,14] and the concentration of which is extremely low in endolymph [5]. Although in some inner ears, the tectorial membrane, otolithic membrane or cupula might assume this role [26], such a role for the cell coat would be of increasing importance in the lizard basilar papilla where there are’regions of hair cells not covered by any overlying structure.
288
Acknowledgements The authors would like to thank Dr. Gustav Engbretson, Institute for Sensory Research, Syracuse University, for providing the lizards and Dr. Alix Robinson, Department of Microbiology, SUNY Upstate Medical Center, for the use of the electron microscope. The help of Kathy Blaine, Pasta Di and Pat Kane is gratefully acknowledged. This research was supported by the Department of Anatomy, SUNY Upstate Medical Center, and NIH Grants. References 1 Belanger. L.F. (1956): Observations on the development, structure and composition of the cochlea of the rat. Ann. Otol. Rhinol. Laryngol. 65, 1060-1073. 2 Bagger-Sjoback, D. and Wersall, J. (1973): The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calotes oersicolor. J. Neurocytol. 2, 329-350. aspects of extracellular 3 Bennett, H.S. (1963): Morphological polysaccharides. J. Histochem. Cytochem. 11, 14-23. 4 Blanquet, P.R. (1976): Ultrahistochemical study on the ruthenium red surface staining. II. Nature and affinity of the electron dense marker. Histochemistry 47, 175-189. 5 Boscher, S.K. and Warren, R.L. (1978): Very low calcium content of cochlear endolymph, an extracellular fluid. Nature (London) 273, 377-378. A.J. (1979): Ionic basis of the 6 Corey, D.P. and Hudspeth. receptor potential in a vertebrate hair cell. Nature (London) 281, 675-677. G.F. (1971): The attachment of the cupulae, 7 Dohlman, otolith and tectorial membrane to the sensory cell areas. Acta Otolaryngol. 71, 89-105. and electrophysio8 Flock. A. (1965): Electron microscopic logical studies on the lateral line canal organ. I. The ultrastructure of the lateral line organ. Acta Otolaryngol. Suppl. 199, l-47. 9 Flock, A., Flock, B. and Murray, E. (1977): Studies on the sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. 83, 85-91. 10 Gulley, R.L. and Reese, T.S. (1977): Regional specializations of the hair cell plasmalemma. Anat. Rec. 189.109-124. basis 11 Hillman, D.E. and Lewis, E.R. (1971): Morphological for a mechanical linkage in otolithic receptor transduction in the frog. Science 174, 416-419. A.J. (1982): Extracellular current flow and the 12 Hudspeth, site of transduction by vertebrate hair cells. J. Neurosci. 2, l-10. 13 Hudspeth, A.J. (1983): Mechanical transduction by hair cells in the acousticolateralis sensory system. Annu. Rev. Neurosci. 6, 187-215. 14 Hudspeth, A.J. and Corey, D.P. (1977): Sensitivity, polar-
15
16
17
18 i9
20
21
22
23
24
25
26
27
28
29
30
ity. and conductance change in the response of vertebrate hair cells to controlled mechanical stimuli. Proc. Natl. ,Acad Sci. U.S.A. 74. 2407-2411. Hudspeth. A.J. and Jacobs, R. (1979): Stereocilia mediate transduction in vertebrate hair cells. Proc. Natl. Acad. SCI. U.S.A. 76. 1506-1509. Kimura. R.S. (1966): Hairs of the cochlear sensory cells and their attachment to the tectorial membrane. Acta Otolaryngol. 61. 55-72. Langer, G.A., Frank. J.S., Nudd. L.M. and Seraydarian, K. (1976): Sialic acid: effect of its removal on calcium exchangeability of cultured heart cells. Science 193. 1013-1015. Luft, J.H. (1971): Ruthenium red and violet. 11. Fme structural localization in animal tissues. Anat. Rec. 171, 369-416. Malcolm, R. (1974): A mechanism by which the hair cells of the inner ear transduce mechanical energy into a modulated train of action potentials. J. Gen. Physiol. 63. 757-772. Mendoza. A.S. and Breipohl, W. (1983): The cell coat of the olfactory epithelium proper and vomeronasal epithelium of the rat as revealed by means of the ruthenium red reaction. Cell Tiss. Res. 230, 1399146. Miller, M.R. (1981): Scanning electron microscope studies of the auditory papilla of some lguanid lizards. Am. J. Anat. 162, 55-72. Moran, D.T.. Rowley, J.C. and Asher, D.L. (1981): Calcium binding sites on sensory processes in vertebrate hair cells, Proc. Natl. Acad. Sci. U.S.A. 78. 3954-3958. Ohmori. H. (1984): Mechanoelectrical transducer has discrete conductances in the chick vestibular hair cell. Proc. Natl. Acad. Sci. U.S.A. 81, 1888-1891. Pickles, J.O., Comis, S.D. and Osborne, M.P. (1984): Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction. Hearing Res. 15, 103-112. Russell, I.J. (1983): Origin of the receptor potential in inner hair cells of the mammalian cochlea - evidence for Davis’ theory. Nature (London) 301, 334-336. Russell, I.J. and Sellick, P.M. (1976): Measurement of potassium and chloride concentrations in the cupulae of the lateral lines of Xenopur faeuis. J. Physiol. 257, 245-255. Sand, 0. (1975): Effects of different ionic environments on the mechano-sensitivity of lateral line organs in the mudpuppy. J. Comp. Physiol. Ser. A 102, 27-42. Slepecky, N. and Chamberlain, S.C. (1982): Distribution and polarity of actin in sensory hair cells of the chinchilla cochlea. Cell Tiss. Res. 224, 15-24. Spoendlin, H. (1968): Ultrastructure and peripheral innervation pattern of the receptor in relation to the first coding of the acoustic message. In: Hearing Mechanisms in Vertebrates, Ciba Foundation Symposium, pp. 89-119. Editors: A.V.S. DeReuck and J. Knight. Churchill, London. Tachibana, M., Saito, H. and Machino, M. (1973): Sulfated acid mucopolysaccharides in the tectorial membrane. Acta Otolaryngol. 76. 37-46.
~fecrrrng Rewarc~h, 17 (1985) 281-288
Elsev1er
HRR 00589
The cell coat of inner ear sensory and supporting ruthenium red * Norma
Slepecky
(Received
’
cells as demonstrated
and Steven C. Chamberlain
21 December
1984: accepted
2X February
by
’
1985)
The ultrastructure of the cell coat of sensory and supporting cells of chinchilla and lizard inner ears was studied using ruthenium red. On the apical surface of both cell types, in both animals, the glycoproteins in the cell coat stain posltwely wth this cationic dye. The apical surface of the sensory hair cells displays no regional variations in cell coat thickness. The uniform stainmg along the length of the stereocilia is not influenced by the normal presence or absence of a tectorial membrane. Although no micro-domains in the glycoproteins that stam positively wth ruthenium red were observed that might correlate with the ultrastructural localization of sites of initiation of the transduction event. the cell coat material on the apical cell surface might play an important role in sequestering ions (particularly calcium) which are required for the transduction process. cochlea.
cell coat, cochlear
hair cell, ruthenium
red
Introduction The surface of almost all cells is covered with a fuzzy layer of mucopolysaccharide, glycoprotein and glycolipid material, associated with and forming a part of, the plasma membrane [3]. Histochemical studies of this layer [18,4] have suggested that the components of the cell coat may be responsible for the way the cell reacts to its external environment. The cell coat may isolate the cell from its surrounding medium and create in effect, a microenvironment. The presence of acidic glycoproteins and glycolipids contributes to a net negative cell surface charge which plays a role in cell-cell and cell-substrate interactions. By virtue of its negative charge, this layer can also sequester cations immediately adjacent to the cell membrane [17]. By limiting or promoting availability of these ions, the cell coat can regulate cell membrane conductance. * All work described in this report was carried out in full compliance with the National Institutes of Health Guidelines for the Use of Experimental Animals. 037%5955/85/$03.30
0 1985 Elsevier Science Publishers
The presence of cell coat material has previously been noted on inner ear cells. The stereocilia of the outer hair cells stain positively for periodate-reactive mucopolysaccharides with the periodic acid Schiff’s (PAS) reaction [l]. Observations with the electron microscope show fibrils surrounding and between adjacent stereocilia [8,2,24]. Cationic probes such as ruthenium red [29] and cationic ferritin [9] stain a prominent cell coat composed of acidic glycoproteins. These findings have led to two suggestions about the function of the surface components of the stereocilia. First, by cross-linking adjacent stereocilia, the cell coat serves to hold the stereocilia together in a bundle so when a stereocilia bundle is deflected, all stereocilia within that bundle move as a group. Second, because of the electronegativity of its acidic groups, the cell coat serves to maintain, by repulsion, a distance between adjacent stereocilia, preventing close contact and preventing the cell membranes from fusing. Recent evidence suggests that stereocilia play an important role in the iransduction process whereby a mechanical stimulus causes depolariza-
B.V. (Biomedical
Division)
282
tion of the hair cell and the generation of a receptor potential. Deflection of the stereocilia initiates this event [15] and at some place along the apical surface of the sensory cell, ion channels specific for positive ions are opened and current flows into the cell [6]. The actual site for this change in membrane conductance has yet to be determined. Initial suggestions based on stereocilia and cuticular plate anatomy included: a deformation-sensitive membrane on the apical cell surface adjacent to the point of insertion of the tallest stereocilia and lying above the basal body and the cuticle-free region of the cuticular plate [ll]; the tapered region of each stereocilium where it pivots when deflected [9]; the lateral surface of the stereocilia where a larger area of ion-permeable membrane would be exposed when stereocilia separate during deflection [19]; and distortion of the cross-links which connect the tips of one row of stereocilia to the adjacent, taller stereocilia in the next row [24]. Recent evidence suggests that the site for changes in the ion conductance mechanism is located at the tips of the stereocilia [12], that all the stereocilia are involved, and that there may be only a few transduction channels per hair cell [13,23,25]. Attempts have been made to localize the site of transduction ultrastructurally, based on the assumptions that such ionic channels might be represented by intramembranous proteins and that their activation requires calcium. Observation and analysis of freeze-fractured stereocilia membranes, however, shows only a random distribution of intramembranous particles. There appear to be no regional specializations along the apical or stereocilia membrane that correspond to proteins that might function as ion channels during transduction [lo]. Attempts to use oxalate or antimonate precipitates to mark sites of calcium-binding proteins have given positive results but the distribution of electron reaction product at specific sites along the apical cell surface is not constant across species [22]. Since the cell coat may also play a role in determining ion conductance, it is of interest to study the cell surface along the apical surface of inner ear cells. Micro-domains of specialized membrane function might be represented by variations of thickness in the cell coat material. For this reason, we have used ruthenium red to char-
acterize the cell coat material and to mark Its distribution along the apical surface of the organ of Corti. In order to preclude any influence of the mucopolysaccharides from the tectorial membrane [7,30] in the study of the cell coat material on the inner and outer hair cell stereocilia, we have also studied the sensory cells from the inner ear of the side-blotched lizard Uta .stansburiana where the cells having stereocilia associated with a tectorial membrane are in a separate region of the basilar papilla from those without a tectorial membrane [211. Materials and methods Inner ears from four chinchillas and four sideblotched lizards (Uta stansburiana) were processed for demonstration of the cell coat using ruthenium red. Those from the same number of animals were processed as controls. Chinchillas were decapitated, the temporal bones removed from the skull and openings made at the apex, round window, and oval window. Care was taken to make sure that Reissner’s membrane was punctured in both the apical and basal regions. Lizards were decapitated, the temporal bones exposed, and openings made in the round and oval windows and in the ampullae of the semicircular canals. Initially, a solution containing 2.5% glutaraldehyde, 0.1 mM MgCl, and 0.125 M phosphate buffer at pH 7.2 was perfused through all openings. Perfusion was continued for 5 min. After this time, temporal bones were placed in scintillation vials containing 10 ml of each subsequent solution, and the vials were placed on a rotator to ensure continuous agitation. Temporal bones were washed in 0.1 mM MgCl, in 0.125 M phosphate buffer at pH 7.2 f 500 pg/ml ruthenium red for 1 h. Then they were postfixed with 2% 0~0, t_ 500 pg/ml ruthenium red in 0.1 mM MgCl, in 0.125 M phosphate buffer at pH 7.2 for 2 h in darkness. Temporal bones were again washed in buffer + ruthenium red for 1 h, then dehydrated through ethanol to propylene oxide and embedded in Araldite. Cochleas were dissected, quarter turns reembedded and trimmed, and thin sections cut and examined unstained with the transmission electron microscope.
283
Results In both the chinchilla and lizard inner ear, sensory and supporting cells display a cell coat which stains positively with ruthenium red. In the chinchilla cochlea, the inner and outer sensory hair cells are located close together on the basilar membrane in the organ of Corti. The stereocilia of both
la
types of hair cells are adjacent to, if not embedded in, the tectorial membrane. In unstained sections of cochleas treated with ruthenium red, it can be seen that the apical surface of the sensory and supporting cells have a uniform layer of electron dense material external to the cell membrane. Outer hair cell stereocilia bundles, cut in crosssection, display this ruthenium red positive
lc
lb Fig. 1. Cross-sections through outer hair cells of the chinchilla cochlea. (la. lb) Electron micrographs of unstained sections of stereo&ha from cochleas processed with ruthenium red. Cell coat material covers the surface of each stereocilium and large accumulations of material connect adjacent stereocilia. (lc) Stereocilia from cochlea processed without ruthenium red. Cell coat material is not obvious and accumulations between adjacent stereocilia are not prominent. Magnifications: la, 24000 x ; lb. 48000 x : lc. 24000x.
284
material around the periphery of each stereocilium (Fig. la, b). In sections which are cut through stereocilia bundles at different points along their length, the coat appears at all levels. Near the tips there are larger accumulations between and connecting adjacent stereocilia. This material can also be observed connecting adjacent stereocilia in cochleas not treated with ruthenium red (Fig. lc), but the material stains prominently when ruthenium red is included in the processing solutions (Fig. 1b). Inner hair cell stereocilia cut in cross section also display a uniform layer of ruthenium red positive material which connects closely apposed stereocilia (Fig. 2a, b). When ruthenium red is omitted from the procedure, the fibrils are not stained and the inner cell stereocilia are separated by a space (Fig. 2~).
When the hair cells are cut longitudinally. the staining pattern along the apical surface of the cell and along the length of the individual stereocilia can be observed (Fig. 3a, c). Stereocilia from both inner and outer hair cells show a thin layer of ruthenium red positive material. There appears to be no variation in the thickness of the cell surface material at the apical surface of the cell, along the lateral membrane of the stereocilium, or at the stereocilium tip (Fig. 3b, d). The cross-bridge linking the tip of the shorter stereocilium with the longer one behind it (Fig. 3d) has been preserved and does not stain prominently with ruthenium red. The staining pattern is similar for the sensory and supporting cells of the basilar papilla of the side-blotched lizard. Two types of hair cells occupy different positions along the papilla. In the
Fig. 2. Cross-sections through inner hair cells of the chinchilla cochlea. (2a, 2b) Electron micrographs of unstained sections of stereocilia from cochleas processed with ruthenium red. Cell coat material covers the surface of each stereocilium and fib&s connect closely apposed stereo&ha - see also Fig. 3a. (2~) Stereocilia from cochlea processed without ruthenium red. Cell coat material is not obvious and stereo&ha appear separate. Magnifications: Za, 12CtOOX ; 2b, 48000 X : SC. 12000 X
2x5
center of the papilla is a region of two to three irregular rows of unidirectionally oriented hair cells with short stereocilia that are covered by a tectorial plate (Fig. 4a). At either end are two rows of bidirectionally oriented hair cells with long stereocilia that lack any association with a tectorial membrane (Fig. 4~). The sensory and supporting cells of the side-blotched lizard basilar papilla also display a cell coat which stains positively with ruthenium red. Again the stereocilia from both
types of hair cells display a uniform material along their lengths (Fig. 4b, d).
layer
of
Discussion The stereocilia of sensory hair cells have been implicated in the transduction process and it is of interest to know where along the apical surface of the sensory hair cells the actual transduction event occurs. It is known that a cell coat is present on
3b
Fig. 3.
Electron
Ruthenium
micrographs
of Ion~tud~naI
red positive material
there is no regional specialization Magnifications:
sections through
forms a uniformly
stereocilia
thick electron-dense
at the tips of stereocilia.
3a. 10200 X ; 3b. 30000 x ; 3c, 10200 x
:
(3a, 3b) Inner
3d, 30000 x
from chinchilla
cochlea
processed with ruthenium
layer on the apical surface of supporting hair cell stereocilia.
(3~. 3d) Outer
red.
and sensory cells:
hair cell stereocilia.
286
the apical surface of inner ear cells. Since the apical surface with its stereo&ha is the region that is thought to undergo ion conductance changes during transduction and since the cell coat is thought to be capable of regulating membrane conductance, further study of the cell coat seems
to be a logical approach to the study of sensory cell function. Histochemical probes such as ruthenium red have been used to characterize the cell coat components of various tissues and specific cell types. Acidic glycoproteins and glycolipids (with carbo-
Fig. 4. Bectron micrographs of sections through the basilar papilla of the side-blotched lizard Uta stamburiunu processed with ruthenium red. (4a) Region of hair cells with short stereocilia and tectorial membrane. (4b) Tips of short stereo&a show no speciakation of ruthenium red positive material. (4c) Region of hair cells with long stereocilia and no tectorial membrane. (4d) Tips of long stereocilia show no regional specialization of ruthenium red positive materiaf. Magnifications: 4a, 800 X : 4b, 30000 X : 4~. soox; 4d, 3oooox.
287
xyl and sulfate groups projecting out from the external surface of the plasma membrane) contribute to a net negative surface charge and it is these groups which are responsible for a ruthenium red positive reaction. Using this cationic dye, it has been determined that the thickness of the cell coat varies in different cells and tissues [18,4]. Within the olfactory system [20] sensory cells of the olfactory epithelium proper stain differently from those of the vomeronasal epithelium, and this difference may be reflected in the differences in odor perception by the two types of receptor organs. In addition, the cell coat of the sensory cells of the vomeronasal epithelium stains differently from that on the adjacent supporting cells. Individual cells may also display regions where thickness of cell coat varies. Capillary endothelial cells have such regions [18] and it is these micro-domains that are thought to correlate with regions of selective permeability. Previous investigators have noted the fibrillar nature of the extracellular material associated with the cells of the inner ear [8,29,7,2] and some have studied its nature and function using histochemical and anionic probes [1,29,9]. In this study we have characterized the cell coat material on inner ear cells using ruthenium red. Both supporting and sensory cells display a cell coat on their external surface. This coat appears of equal thickness on both cell types and also on inner and outer hair cell apical surfaces and stereocilia. Near the tips of the outer hair cell stereocilia large accumulations of ruthenium red positive-staining material are found connecting adjacent stereocilia within a bundle. When cochleas are processed with ruthenium red in the solutions, stereocilia of inner hair cells are connected by fibrils and are seen in close apposition, similar to their configuration when cochleas are processed for scanning electron microscopy. They appear separate in cochleas processed in the absence of ruthenium red (Fig. 2c; [16]). Since stereocilia are known to taper near their point of insertion into the cuticular plate, this observation must be treated with caution and the interpretation depends on the plane of section. In the specimens examined, inner hair cell stereocilia had been cross-sectioned close to the tips of the longest stereocilia, and not close to the region of
the taper. The apposition was also observed when stereocilia were sectioned longitudinally as shown in Fig. 3a and b. It is not yet known if this is because ruthenium red preserves the interactions at the tips of adjacent inner hair cell stereocilia, or if by neutralizing the electronegativity on the surface, it allows the stereocilia to move closer together than is normal. In either case, it may be another indication that the stereocilia of inner hair cells are different from the stereocilia of outer hair cells [28]. The stereocilia of both chinchilla and lizard show no differences in cell coat staining along their length which might indicate a region specialized for the initiation of the transduction event. Although others have had similar results when analyzing intramembranous particles [lo] and localizing calcium binding proteins [22], this does not prove that micro-domains in the cell surface do not exist. Pickles et al. [24] have preserved some of the cross-linking material that links the tips of the shorter stereocilia with the longer stereocilia behind them, by using glutaraldehyde without osmium postfixation, and have suggested that the stretching of these cross-bridges might deform the stereocilia membrane and initiate the transduction event. This material does not appear to be stained prominently with ruthenium red, so its components may be other than acidic glycoproteins. Other histochemical probes may prove useful in characterizing and localizing this material. As the ruthenium red-positive material appears to be distributed along the apical surface of the sensory and supporting cells regardless of their position with respect to the tectorial membrane, the cell surface may still play a role in transduction. Since acidic glyco-conjugates along the cell surface can sequester cations, it might be expected that the cell coat along the apical surface of these cells might create a cation-rich micro-environment [17] - particularly for calcium - an ion which is necessary for transduction [27,14] and the concentration of which is extremely low in endolymph [5]. Although in some inner ears, the tectorial membrane, otolithic membrane or cupula might assume this role [26], such a role for the cell coat would be of increasing importance in the lizard basilar papilla where there are’regions of hair cells not covered by any overlying structure.
288
Acknowledgements The authors would like to thank Dr. Gustav Engbretson, Institute for Sensory Research, Syracuse University, for providing the lizards and Dr. Alix Robinson, Department of Microbiology, SUNY Upstate Medical Center, for the use of the electron microscope. The help of Kathy Blaine, Pasta Di and Pat Kane is gratefully acknowledged. This research was supported by the Department of Anatomy, SUNY Upstate Medical Center, and NIH Grants. References 1 Belanger. L.F. (1956): Observations on the development, structure and composition of the cochlea of the rat. Ann. Otol. Rhinol. Laryngol. 65, 1060-1073. 2 Bagger-Sjoback, D. and Wersall, J. (1973): The sensory hairs and tectorial membrane of the basilar papilla in the lizard Calotes oersicolor. J. Neurocytol. 2, 329-350. aspects of extracellular 3 Bennett, H.S. (1963): Morphological polysaccharides. J. Histochem. Cytochem. 11, 14-23. 4 Blanquet, P.R. (1976): Ultrahistochemical study on the ruthenium red surface staining. II. Nature and affinity of the electron dense marker. Histochemistry 47, 175-189. 5 Boscher, S.K. and Warren, R.L. (1978): Very low calcium content of cochlear endolymph, an extracellular fluid. Nature (London) 273, 377-378. A.J. (1979): Ionic basis of the 6 Corey, D.P. and Hudspeth. receptor potential in a vertebrate hair cell. Nature (London) 281, 675-677. G.F. (1971): The attachment of the cupulae, 7 Dohlman, otolith and tectorial membrane to the sensory cell areas. Acta Otolaryngol. 71, 89-105. and electrophysio8 Flock. A. (1965): Electron microscopic logical studies on the lateral line canal organ. I. The ultrastructure of the lateral line organ. Acta Otolaryngol. Suppl. 199, l-47. 9 Flock, A., Flock, B. and Murray, E. (1977): Studies on the sensory hairs of receptor cells in the inner ear. Acta Otolaryngol. 83, 85-91. 10 Gulley, R.L. and Reese, T.S. (1977): Regional specializations of the hair cell plasmalemma. Anat. Rec. 189.109-124. basis 11 Hillman, D.E. and Lewis, E.R. (1971): Morphological for a mechanical linkage in otolithic receptor transduction in the frog. Science 174, 416-419. A.J. (1982): Extracellular current flow and the 12 Hudspeth, site of transduction by vertebrate hair cells. J. Neurosci. 2, l-10. 13 Hudspeth, A.J. (1983): Mechanical transduction by hair cells in the acousticolateralis sensory system. Annu. Rev. Neurosci. 6, 187-215. 14 Hudspeth, A.J. and Corey, D.P. (1977): Sensitivity, polar-
15
16
17
18 i9
20
21
22
23
24
25
26
27
28
29
30
ity. and conductance change in the response of vertebrate hair cells to controlled mechanical stimuli. Proc. Natl. ,Acad Sci. U.S.A. 74. 2407-2411. Hudspeth. A.J. and Jacobs, R. (1979): Stereocilia mediate transduction in vertebrate hair cells. Proc. Natl. Acad. SCI. U.S.A. 76. 1506-1509. Kimura. R.S. (1966): Hairs of the cochlear sensory cells and their attachment to the tectorial membrane. Acta Otolaryngol. 61. 55-72. Langer, G.A., Frank. J.S., Nudd. L.M. and Seraydarian, K. (1976): Sialic acid: effect of its removal on calcium exchangeability of cultured heart cells. Science 193. 1013-1015. Luft, J.H. (1971): Ruthenium red and violet. 11. Fme structural localization in animal tissues. Anat. Rec. 171, 369-416. Malcolm, R. (1974): A mechanism by which the hair cells of the inner ear transduce mechanical energy into a modulated train of action potentials. J. Gen. Physiol. 63. 757-772. Mendoza. A.S. and Breipohl, W. (1983): The cell coat of the olfactory epithelium proper and vomeronasal epithelium of the rat as revealed by means of the ruthenium red reaction. Cell Tiss. Res. 230, 1399146. Miller, M.R. (1981): Scanning electron microscope studies of the auditory papilla of some lguanid lizards. Am. J. Anat. 162, 55-72. Moran, D.T.. Rowley, J.C. and Asher, D.L. (1981): Calcium binding sites on sensory processes in vertebrate hair cells, Proc. Natl. Acad. Sci. U.S.A. 78. 3954-3958. Ohmori. H. (1984): Mechanoelectrical transducer has discrete conductances in the chick vestibular hair cell. Proc. Natl. Acad. Sci. U.S.A. 81, 1888-1891. Pickles, J.O., Comis, S.D. and Osborne, M.P. (1984): Cross-links between stereocilia in the guinea pig organ of Corti, and their possible relation to sensory transduction. Hearing Res. 15, 103-112. Russell, I.J. (1983): Origin of the receptor potential in inner hair cells of the mammalian cochlea - evidence for Davis’ theory. Nature (London) 301, 334-336. Russell, I.J. and Sellick, P.M. (1976): Measurement of potassium and chloride concentrations in the cupulae of the lateral lines of Xenopur faeuis. J. Physiol. 257, 245-255. Sand, 0. (1975): Effects of different ionic environments on the mechano-sensitivity of lateral line organs in the mudpuppy. J. Comp. Physiol. Ser. A 102, 27-42. Slepecky, N. and Chamberlain, S.C. (1982): Distribution and polarity of actin in sensory hair cells of the chinchilla cochlea. Cell Tiss. Res. 224, 15-24. Spoendlin, H. (1968): Ultrastructure and peripheral innervation pattern of the receptor in relation to the first coding of the acoustic message. In: Hearing Mechanisms in Vertebrates, Ciba Foundation Symposium, pp. 89-119. Editors: A.V.S. DeReuck and J. Knight. Churchill, London. Tachibana, M., Saito, H. and Machino, M. (1973): Sulfated acid mucopolysaccharides in the tectorial membrane. Acta Otolaryngol. 76. 37-46.