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Gap junction systems in the mammalian cochlea
Brain Research Reviews 32 Ž2000. 163–166 www.elsevier.comrlocaterbres
Short review
Gap junction systems in the mammalian cochlea Toshihiko Kikuchi a
a,c,)
, Robert S. Kimura a , David L. Paul b, Tomonori Takasaka c , Joe C. Adams a
Department of Otolaryngology, HarÕard Medical School and Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA b Department of Neurobiology, HarÕard Medical School, 220 Longwood AÕenue, Boston, MA 02115, USA c Department of Otolaryngology, Tohoku UniÕersity School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
Abstract Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of Kq ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of Kq and result in hearing loss. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Connexin 26; Kq; Ion transport
Contents 1. Introduction .
.......................................................................
2. Distribution of connexin 26 in the cochlea . 3. Epithelial cell gap junction system .
.......................................................
164
...........................................................
165
4. Connective tissue cell gap junction system .
.......................................................
5. Functional significance of gap junction systems in the ion transport mechanism .
165
...................................
166
.....................................................................
166
..........................................................................
166
Acknowledgements . References
164
) Corresponding author. Department of Otolaryngology, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980-8574, Japan. Fax: q81-22-717-7307; e-mail: [email protected]
0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 7 6 - 4
164
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
1. Introduction The mammalian cochlea has a unique arrangement of extracellular fluid-filled compartments that support the mechanosensory transduction process. The apical surfaces of the cochlear hair cells are bathed in endolymph, which has an ionic composition of high Kq and low Naq. The cochlear endolymph is also characterized by the presence of the endolymphatic potential, of approximately 100 mV. When hair cells are activated by sound, their receptor potentials are generated by the flow of Kq from endolymph into hair cells. The Kq is then released to extracellular space and from here is recirculated back to endolymph. The gap junction systems are most likely the pathway for the recirculation of cochlear Kq w5x. Recent molecular biological studies w2,3x have shown that mutation in connexin 26 gene can cause non-syn-
dromic sensorineural hearing loss, and also strongly support our hypothesis regarding the role of gap junctions in mechanisms of the inner ear.
2. Distribution of connexin 26 in the cochlea Immunohistochemical localization of connexin 26 at the light microscopic and ultrastructural levels in the rat inner ear was studied in detail w4,5x. We have since confirmed the immunostaining results in mouse, gerbil, guinea pig, chinchilla, cat, and human, and believe that the initial results are representative of a general organization of mammalian ears. The mammalian cochlea is comprised of a variety of cell components, including various epithelial and connective tissue cells. Connexin 26-like immunoreactivity was
Fig. 1. Ža. Rat cochlear lateral wall immunostained with anti-connexin 26 antibody. Intense immunoreactivity is observed among fibrocytes in the spiral ligament and along the basal cells of the stria vascularis. No connexin 26-like immunoreactivity is observed in the spiral prominence epithelium Žarrows.. R, Reissner’s membrane; SSZ, suprastrial zone; StV, stria vascularis; I, type I fibrocyte area; II, type II fibrocyte area. Bar s 10 mm. Žb. Connexin 26-like immunoreactivity is present among the cochlear supporting cells. IHC, inner hair cell; OHC, outer hair cell; TM, tectorial membrane. Bar s 5 mm. Žc. Spiral limbus at the basal turn. Connexin 26-like immunoreactivity is observed among the connective tissue cells. SG, Spiral ganglion. Bars 10 mm. Žd. Spiral limbus at the apical turn. Connexin 26 exists among the fibrocytes. Some fibrocytes occasionally extend downward Žarrows. and reach the scala tympani. Bar s 10 mm.
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
observed among the basal cells of the stria vascularis, in the types I and II fibrocyte areas of the spiral ligament, in the suprastrial zone ŽFig. 1a., and in the spiral limbus ŽFig. 1c,d.. In the organ of Corti, connexin 26 immunostaining was observed among supporting cells ŽFig. 1b.. In contrast, the spiral prominence epithelium, the strial marginal cell layer and Reissner’s membrane were not immunostained. Immunohistochemical localization of connexin 26 corresponded well to the distribution pattern of gap junctions visualized by transmission electron microscopy w5x. These results indicate connexin 26 immunostaining provides a reasonable overview of the locations of gap junctions in the cochlea.
3. Epithelial cell gap junction system All cochlear supporting cells are directly connected to adjacent supporting cells with gap junctions. No definitive gap junctions or connexin 26-like immunoreactivity is detected between the cochlear sensory cells and the supporting cells. In the lateral end of the cochlear supporting cells, gap junctions are observed between Claudius cells and root cells. The root cells extend their cytoplasmic processes, which are also connected by gap junctions, deep into the lower part of the spiral ligament, and a large number of type II fibrocytes encircles these root processes. No direct contact is found between root cells and type II fibrocytes. On the medial side of the organ of Corti, inner sulcus cells, which are the innermost supporting cells, are connected by gap junctions to interdental cells. These findings suggest the presence of gap junctional communication among the cochlear supporting cells ex-
165
tending from root cells to interdental cells, and thereby forming a large gap junctional network, the epithelial cell gap junction system. In contrast, gap junctional communication is not found between the hair cells and supporting cells. At its proximal and distal extremes, the epithelial cell gap junction system is separated from adjacent connective tissue cells by a continuous basement membrane.
4. Connective tissue cell gap junction system Connective tissue cells in the spiral limbus are coupled to each other and make a large gap junctional network. Mesenchymal cells, which line the scala vestibuli, possess scattered gap junctions, and also have gap junctional connections with fibrocytes in the spiral limbus and with the fibrocytes in the suprastrial zone of the spiral ligament. Fibrocytes in the spiral ligament are classified into the four different type of cells w5,7x. They are composed of types I, II, III, and IV fibrocytes. All fibrocytes within the cochlear lateral wall are coupled via gap junctions. The stria vascularis is made up of three different type of cells, including marginal cells, intermediate cells, and basal cells. The strial basal cells have gap junctional communication with strial intermediate cells and with fibrocytes in the spiral ligament. However, marginal cells are excluded from this gap junctional network. These connections via gap junctions demonstrate that strial intermediate and basal cells, four type of fibrocytes in the cochlear lateral wall, mesenchymal cells lining the bony otic capsule of the scala vestibuli, and connective tissue cells in the spiral limbus constitute an extensive gap
Fig. 2. A schematic illustration indicating the possible pathway for the transport of Kq in the cochlea.
166
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
junctional network. We refer to this system as the connective tissue cell gap junction system.
5. Functional significance of gap junction systems in the ion transport mechanism It is well-established that the high level of Kq in the scala media that is necessary for normal functioning of hair cells is maintained by the activity of marginal cells of the stria vascularis. The marginal cells accumulate Kq using a combination of Na,K-ATPase and Na–K–Cl cotransporter found in their basolateral plasma membranes w8x. It has been established that the Kq accumulated by the stria vascularis come from the perilymphatic space, but Kq in perilymph are prevented direct to access to the intrastrial space by a network of tight junctions which is present among the basal cells of the stria vascularis. Within the spiral ligament there are cells called type II fibrocytes that have the same specialization as strial marginal cells for accumulating Kq. Our data indicate that Kq taken up by intense Na,K-ATPase w6,7x and Na–K–Cl cotransporter w1x within the plasma membrane of type II fibrocytes enters the stria vascularis via a system of gap junctions that connects type II fibrocytes to the intrastrial space ŽFig. 2.. The Kq taken up by the strial marginal cells are expelled into the scala media, where they serve as the dominant cation which carries hair cell receptor currents. After entering the sensory hair cells in response to mechanical vibration of the cochlea the Kq is expelled basolaterally by sensory hair cells and appears to be delivered back to the type II fibrocytes via the epithelial cell gap junction system.
Acknowledgements This work was supported by Research Grant No. 10671581 from the Ministry of Education, Science and Culture, Japan and NIDCDrNIH Grant R01 DC00073.
References w1x J.J. Crouch, N. Sakaguchi, C. Lytle, B.A. Schulte, Immunohistochemical localization of the Na–K–Cl co-transporter ŽNKCC1. in the gerbil inner ear, J. Histochem. Cytochem. 45 Ž1997. 773–778. w2x X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, E. Mansfield, E. Rappaport, N. Govea, M. Mila, L. Zelante, P. Gasparini, Connexin-26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 Ž1998. 394–398. w3x D.P. Kelsell, J. Dunlop, H.P. Stevens, N.J. Lench, J.N. Liang, G. Parry, R.F. Mueller, I.M. Leigh, Connexin 26 mutations in hereditary non-syndromic sensorineural deafness, Nature 387 Ž1997. 80–83. w4x T. Kikuchi, J.C. Adams, D.L. Paul, R.S. Kimura, Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis, Acta Otolaryngol. ŽStockholm. 114 Ž1994. 520–528. w5x T. Kikuchi, R.S. Kimura, D.L. Paul, J.C. Adams, Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis, Anat. Embryol. 191 Ž1995. 101–118. w6x B.A. Schulte, J.C. Adams, Distribution of immunoreactive Naq,KqATPase in the gerbil cochlea, J. Histochem. Cytochem. 7 Ž1989. 127–134. w7x S.S. Spicer, B.A. Schulte, Differentiation of inner ear fibrocytes according to their ionic transport related activity, Hear. Res. 56 Ž1991. 53–64. w8x P. Wangemann, Comparison of ion transport mechanisms between vestibular dark cells and strial marginal cells, Hear. Res. 90 Ž1995. 149–157.
Short review
Gap junction systems in the mammalian cochlea Toshihiko Kikuchi a
a,c,)
, Robert S. Kimura a , David L. Paul b, Tomonori Takasaka c , Joe C. Adams a
Department of Otolaryngology, HarÕard Medical School and Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, USA b Department of Neurobiology, HarÕard Medical School, 220 Longwood AÕenue, Boston, MA 02115, USA c Department of Otolaryngology, Tohoku UniÕersity School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan
Abstract Recent findings that a high proportion of non-syndromic hereditary sensorineural hearing loss is due to mutations in the gene for connexin 26 indicate the crucial role that the gene product plays for normal functioning of the cochlea. Excluding sensory cells, most cells in the cochlea are connected via gap junctions and these gap junctions appear to play critical roles in cochlear ion homeostasis. Connexin 26 occurs in gap junctions connecting all cell classes in the cochlea. There are two independent systems of cells, which are defined by interconnecting gap junctions. The first system, the epithelial cell gap junction system, is mainly composed of all organ of Corti supporting cells, and also includes interdental cells in the spiral limbus and root cells within the spiral ligament. The second system, the connective tissue cell gap junction system, consists of strial intermediate cells, strial basal cells, fibrocytes in the spiral ligament, mesenchymal cells lining the bony otic capsule facing the scala vestibuli, mesenchymal dark cells in the supralimbal zone, and fibrocytes in the spiral limbus. One function of these gap junctional systems is the recirculation of Kq ions from hair cells to the strial marginal cells. Interruption of this recirculation, which may be caused by the mutation in connexin 26 gene, would deprive the stria vascularis of Kq and result in hearing loss. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Connexin 26; Kq; Ion transport
Contents 1. Introduction .
.......................................................................
2. Distribution of connexin 26 in the cochlea . 3. Epithelial cell gap junction system .
.......................................................
164
...........................................................
165
4. Connective tissue cell gap junction system .
.......................................................
5. Functional significance of gap junction systems in the ion transport mechanism .
165
...................................
166
.....................................................................
166
..........................................................................
166
Acknowledgements . References
164
) Corresponding author. Department of Otolaryngology, Tohoku University School of Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980-8574, Japan. Fax: q81-22-717-7307; e-mail: [email protected]
0165-0173r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 1 7 3 Ž 9 9 . 0 0 0 7 6 - 4
164
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
1. Introduction The mammalian cochlea has a unique arrangement of extracellular fluid-filled compartments that support the mechanosensory transduction process. The apical surfaces of the cochlear hair cells are bathed in endolymph, which has an ionic composition of high Kq and low Naq. The cochlear endolymph is also characterized by the presence of the endolymphatic potential, of approximately 100 mV. When hair cells are activated by sound, their receptor potentials are generated by the flow of Kq from endolymph into hair cells. The Kq is then released to extracellular space and from here is recirculated back to endolymph. The gap junction systems are most likely the pathway for the recirculation of cochlear Kq w5x. Recent molecular biological studies w2,3x have shown that mutation in connexin 26 gene can cause non-syn-
dromic sensorineural hearing loss, and also strongly support our hypothesis regarding the role of gap junctions in mechanisms of the inner ear.
2. Distribution of connexin 26 in the cochlea Immunohistochemical localization of connexin 26 at the light microscopic and ultrastructural levels in the rat inner ear was studied in detail w4,5x. We have since confirmed the immunostaining results in mouse, gerbil, guinea pig, chinchilla, cat, and human, and believe that the initial results are representative of a general organization of mammalian ears. The mammalian cochlea is comprised of a variety of cell components, including various epithelial and connective tissue cells. Connexin 26-like immunoreactivity was
Fig. 1. Ža. Rat cochlear lateral wall immunostained with anti-connexin 26 antibody. Intense immunoreactivity is observed among fibrocytes in the spiral ligament and along the basal cells of the stria vascularis. No connexin 26-like immunoreactivity is observed in the spiral prominence epithelium Žarrows.. R, Reissner’s membrane; SSZ, suprastrial zone; StV, stria vascularis; I, type I fibrocyte area; II, type II fibrocyte area. Bar s 10 mm. Žb. Connexin 26-like immunoreactivity is present among the cochlear supporting cells. IHC, inner hair cell; OHC, outer hair cell; TM, tectorial membrane. Bar s 5 mm. Žc. Spiral limbus at the basal turn. Connexin 26-like immunoreactivity is observed among the connective tissue cells. SG, Spiral ganglion. Bars 10 mm. Žd. Spiral limbus at the apical turn. Connexin 26 exists among the fibrocytes. Some fibrocytes occasionally extend downward Žarrows. and reach the scala tympani. Bar s 10 mm.
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
observed among the basal cells of the stria vascularis, in the types I and II fibrocyte areas of the spiral ligament, in the suprastrial zone ŽFig. 1a., and in the spiral limbus ŽFig. 1c,d.. In the organ of Corti, connexin 26 immunostaining was observed among supporting cells ŽFig. 1b.. In contrast, the spiral prominence epithelium, the strial marginal cell layer and Reissner’s membrane were not immunostained. Immunohistochemical localization of connexin 26 corresponded well to the distribution pattern of gap junctions visualized by transmission electron microscopy w5x. These results indicate connexin 26 immunostaining provides a reasonable overview of the locations of gap junctions in the cochlea.
3. Epithelial cell gap junction system All cochlear supporting cells are directly connected to adjacent supporting cells with gap junctions. No definitive gap junctions or connexin 26-like immunoreactivity is detected between the cochlear sensory cells and the supporting cells. In the lateral end of the cochlear supporting cells, gap junctions are observed between Claudius cells and root cells. The root cells extend their cytoplasmic processes, which are also connected by gap junctions, deep into the lower part of the spiral ligament, and a large number of type II fibrocytes encircles these root processes. No direct contact is found between root cells and type II fibrocytes. On the medial side of the organ of Corti, inner sulcus cells, which are the innermost supporting cells, are connected by gap junctions to interdental cells. These findings suggest the presence of gap junctional communication among the cochlear supporting cells ex-
165
tending from root cells to interdental cells, and thereby forming a large gap junctional network, the epithelial cell gap junction system. In contrast, gap junctional communication is not found between the hair cells and supporting cells. At its proximal and distal extremes, the epithelial cell gap junction system is separated from adjacent connective tissue cells by a continuous basement membrane.
4. Connective tissue cell gap junction system Connective tissue cells in the spiral limbus are coupled to each other and make a large gap junctional network. Mesenchymal cells, which line the scala vestibuli, possess scattered gap junctions, and also have gap junctional connections with fibrocytes in the spiral limbus and with the fibrocytes in the suprastrial zone of the spiral ligament. Fibrocytes in the spiral ligament are classified into the four different type of cells w5,7x. They are composed of types I, II, III, and IV fibrocytes. All fibrocytes within the cochlear lateral wall are coupled via gap junctions. The stria vascularis is made up of three different type of cells, including marginal cells, intermediate cells, and basal cells. The strial basal cells have gap junctional communication with strial intermediate cells and with fibrocytes in the spiral ligament. However, marginal cells are excluded from this gap junctional network. These connections via gap junctions demonstrate that strial intermediate and basal cells, four type of fibrocytes in the cochlear lateral wall, mesenchymal cells lining the bony otic capsule of the scala vestibuli, and connective tissue cells in the spiral limbus constitute an extensive gap
Fig. 2. A schematic illustration indicating the possible pathway for the transport of Kq in the cochlea.
166
T. Kikuchi et al.r Brain Research ReÕiews 32 (2000) 163–166
junctional network. We refer to this system as the connective tissue cell gap junction system.
5. Functional significance of gap junction systems in the ion transport mechanism It is well-established that the high level of Kq in the scala media that is necessary for normal functioning of hair cells is maintained by the activity of marginal cells of the stria vascularis. The marginal cells accumulate Kq using a combination of Na,K-ATPase and Na–K–Cl cotransporter found in their basolateral plasma membranes w8x. It has been established that the Kq accumulated by the stria vascularis come from the perilymphatic space, but Kq in perilymph are prevented direct to access to the intrastrial space by a network of tight junctions which is present among the basal cells of the stria vascularis. Within the spiral ligament there are cells called type II fibrocytes that have the same specialization as strial marginal cells for accumulating Kq. Our data indicate that Kq taken up by intense Na,K-ATPase w6,7x and Na–K–Cl cotransporter w1x within the plasma membrane of type II fibrocytes enters the stria vascularis via a system of gap junctions that connects type II fibrocytes to the intrastrial space ŽFig. 2.. The Kq taken up by the strial marginal cells are expelled into the scala media, where they serve as the dominant cation which carries hair cell receptor currents. After entering the sensory hair cells in response to mechanical vibration of the cochlea the Kq is expelled basolaterally by sensory hair cells and appears to be delivered back to the type II fibrocytes via the epithelial cell gap junction system.
Acknowledgements This work was supported by Research Grant No. 10671581 from the Ministry of Education, Science and Culture, Japan and NIDCDrNIH Grant R01 DC00073.
References w1x J.J. Crouch, N. Sakaguchi, C. Lytle, B.A. Schulte, Immunohistochemical localization of the Na–K–Cl co-transporter ŽNKCC1. in the gerbil inner ear, J. Histochem. Cytochem. 45 Ž1997. 773–778. w2x X. Estivill, P. Fortina, S. Surrey, R. Rabionet, S. Melchionda, L. D’Agruma, E. Mansfield, E. Rappaport, N. Govea, M. Mila, L. Zelante, P. Gasparini, Connexin-26 mutations in sporadic and inherited sensorineural deafness, Lancet 351 Ž1998. 394–398. w3x D.P. Kelsell, J. Dunlop, H.P. Stevens, N.J. Lench, J.N. Liang, G. Parry, R.F. Mueller, I.M. Leigh, Connexin 26 mutations in hereditary non-syndromic sensorineural deafness, Nature 387 Ž1997. 80–83. w4x T. Kikuchi, J.C. Adams, D.L. Paul, R.S. Kimura, Gap junction systems in the rat vestibular labyrinth: immunohistochemical and ultrastructural analysis, Acta Otolaryngol. ŽStockholm. 114 Ž1994. 520–528. w5x T. Kikuchi, R.S. Kimura, D.L. Paul, J.C. Adams, Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis, Anat. Embryol. 191 Ž1995. 101–118. w6x B.A. Schulte, J.C. Adams, Distribution of immunoreactive Naq,KqATPase in the gerbil cochlea, J. Histochem. Cytochem. 7 Ž1989. 127–134. w7x S.S. Spicer, B.A. Schulte, Differentiation of inner ear fibrocytes according to their ionic transport related activity, Hear. Res. 56 Ž1991. 53–64. w8x P. Wangemann, Comparison of ion transport mechanisms between vestibular dark cells and strial marginal cells, Hear. Res. 90 Ž1995. 149–157.