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Observations on Morphological Features and Mechanical Properties of the Peripheral Vestibular Receptor System in the Frog
69
Observations on Morphological Features and Mechanical Properties of the Peripheral Vestibular Receptor System in the Frog D. E. H I L L M A N
Division of Neurobiolofy, Department of Physiology and Biophysics, University of Iowa, Iowa City, l o wa, U S . A
.
Breuer (1891) noted that tuft-like structures projected from the surface of the receptor epithelium. He attributed the receptor function to the shearing motion of' these hairlike masses. With electron microscopy two types of cilia are found in the tuft (Wersall, 1956): (i) numerous stereocilia which are positioned over a relatively ridged cuticular plate that is placed at the luminal end of the cell and (ii) a single kinocilium which stands over a notch in this plate (Flock and Wersall, 1962; Spoendlin, 1965; Hillman, 1969) (Figs. 1, 2). Since all the kinocilia are on the same side of the tuft Lowenstein and Wersall (1959) were able to relate this morphological polarity to the activity within the vestibular nerve. A force which was directed toward the kinocilium caused an increase in the activity of the vestibular nerve while a decrease in activity was seen when the force was directed away from the kinociliary side of the cell. The action of the ciliary hairs in the otolithic organs following a positional change shows that the sterocilia have a sliding motion at their distal ends (Hillman and Lewis, 1971) (Fig. 3B-D). The kinocilia, however, are different in that the distal portion of the kinocilium, at least in the case of the frog, is attached to the adjacent stereocilia (Figs. 2, 3A). For this reason the kinocilium cannot slide at its distal end but is displaced at its base (Figs. 2,4). When a force is directed toward the kinociliary side of the cell, a plunging motion results on the kinocilium which produces a deformation of the cell membrane at the kinociliary base (Fig. 4A, B). At the same time a distension of the remaining membrane over the cuticular notch occurs as a protrusion of the cell in the notch region (Fig. 4A, B). When a force is applied in the opposite direction (toward the stereociliary side of the cell) the base of the kinocilium is raised while the stereocilia, which stand on the ridged cuticle, slide at their distal ends (Figs. 3B, 4C, D). This results in a rounding of the surface membrane of the cell over the notch region and thereby reduces the distension of the membrane at this active site (Fig. 4C, D). The result is a directional mechanism which is postulated to produce the conductive changes necessary for modulation of transmitter release and the activation of afferent fibers. The relationship between the ciliary apparatus and the otolithic membrane is compatible with the plunging motion of the kinocilium. In the case of the sacculus in References p . 75
70
D.E. H I L L M A N
Fig. 1 . Scanning electron micrograph showing the ciliary tufts on the saccular macula of the frog. The ciliary tuft is composed of stereocilia (S) and a single kinocilium (K) which has a bulb at its apex. The ciliary tufts are bent away from the kinociliary side of the cell. A filamentous mat (FB) which is over the supporting cells delineates the luminal surface of the receptor cells.
PERIPHERAL VESTIBULAR RECEPTOR SYSTEM IN FROG
71
Fig. 2. Diagram to show the vestibular ciliary apparatus and its relationship to the apical end of the receptor cell in the frog. The stereocilia (S) stand on the cuticular plate (C) which is in the apical end of the receptor cell. The kinocilium (K) is positioned over a notch (N) in the cuticle where its base is in contact with the cell cytoplasm. Due to a filamentous attachment of the kinociliary bulb to adjacent stereocilia the pliable region in the area of the cuticular notch can yield to axial motion of the kinocilium. Two rows of stereocilia are cut at their base.
the frog, the bulb at the apex of the kinocilium (Figs. 1-3) is attached to the rim of a port in the otolithic membrane (Hillman and Lewis, 1971). In the utricle where the kinocilium is 3 to 4 times longer than the longest stereocilia the apical part of the kinocilium is embedded in the otolithic membrane. The otolithic membrane is attached to supporting cells by filament-like structures (Figs. 1, 3B-D) which cross the submembranous base to become part of the otolithic membrane (Hillman and Lewis, 1971). This submembranous zone is the shearing site for displacement between the supporting cells and the otolithic membranes (Hillman, in preparation). A displacement of the otolithic membrane draws the kinocilium and the adjacent stereocilia in the direction of its motion and thereby produces the axial motion in the kinociliary shaft which is related to the direction of the motion (Fig. 4). In the canal system the ciliary processes are 50 ,um or more in length. A row of stereocilia, 5 or 6, accompanies the kinocilium across the subcupular space where the kinocilium enters and is embedded in the cupula. Like the otolithic membrane, the subcupular space is crossed by fine filaments which anchor the cupuIa to the supporting cell (Dohlman, 1971). The action of the cupula is the subject of much controversy since varied opinions References p . 75
72
D. E. H I L L M A N
and observations exist (Steinhausen, 1934; Trincker, 1962; Zalin, 1967; Dohlman, 1969). It has been commonly accepted that the cupula acts as a swinging door with its axis on the crista. Numerous factors, however, indicate that such a movement is incompatible with the properties of such a structure. An alternative possibility is an elastic diaphragm which has a resiliency that would return it to the original position. The small displacement volume which Oman and Young (1972) calculated for physiological movement indicates that under the conditions of their study, the movement of the hairs would be negligible. An elastic diaphragm which is bound circumferentially to the ampullary wall, except for the crista where it is loosely bound by filaments which cross the wide subcupular space, would have its greatest displacement near the crista somewhat like the pendulum proposed by Zalin (1967) (Fig. 4E). This would result in the greatest amount of ciliary displacement for a given volume of endolymph. The cupular zone near the center of the crista would have minimal resistance and could allow for responses to small movements. On the other hand, during large endolymph changes (possibly non-physiological), attachment of a cupula to the crista through the kinocilia and filaments holds the cupular base to this structure while the apex breaks away (Hillman and Llinas, unpublished observation) and thereby gives the classical swinging door appearance seen by Steinhausen (1934), Dohlman (1969) and other investigators. To amplify the small movement which appears to be present during small angular deviations, the ciliary apparatus itself may bend in an arching manner which physically amplifies the axial motion of the kinocilium over and above that o f straight processes (Fig. 4F). This arching may be due in part to the pipe organ arrangement of the stereocilia which have a graded resistance so that bending away from the kinociliuni produces a pulley-like arch for the kinocilium (Fig. 3B). With motion toward the kinociliary side of the cell, the longer stereocilia have the least resistance so that bending occurs near the distal end of the kinocilium as a distal arch of the ciliary apparatus. Arching which results over a small length of the kinocilium can greatly enhance the displacement seen at the base of the kinocilium. In a model of a 50 pm long kino-stereociliary tuft which has a contoured bend the displacement at the base as significant with a 3 pm apical displacement (Fig. 4F). This niovement is in the realm
Fig. 3 . Transmission and scanning electron micrographs from the frog to show the relationship between the stereocilia and kinocilium due to displacement during fixation of the sacculus. A, Transmission electron micrograph showing the filaments which bind the apical end of the kinocilium (K) to an adjacent stereocilium (S). Small filamentous processes arise from a dense zone on the kinociliary membrane and traverse a cleft to join the membrane of the stereocilium. B, Scanning electron micrograph showing the ciliary tuft of the kinocilium (K) and numerous stereocilia which are bent away from the kinociliary side of the receptor cell (RC). A filamentous mat (FB) overlies the supporting cells. C , Scanning electron micrograph from the region of the striola of the saccular macula showing the opposed orientation of the ciliary tufts. D, A scanning electron micrograph showing two directionally opposed ciliary tufts which have been fixed while the gravitational force on the otolithic mass was changed by 180". The top stereociliary tuft is bent away from the kinociliary side of the cell while the lower tuft is bent toward the kinociliary side of the cell. The sliding between the stereocilia is obvious in this preparation. At the arrow the raised cell membrane over the cuticular notch is evident. B and D from Hillman and Lewis (1971).
P E R I P H E R A L V E S T I B U L A R R E C E P T O R SYSTEM I N F R O G
References p. 75
73
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D. E. H I L L M A N
A
C
E
Fig. 4. Diagrams and transmission electron micrographs to show the axial motion of the kinocilium due to directional displacement of the ciliary tuft. A and B, Motion toward the kinociliary side of the cell causes the kinocilium (K)to plunge into the cuticular notch (N) which results in a deformation and distention of the membrane in this region. C and D, Displacement away from the kinociliary side of the cell raises the base of the kinocilium causing the membrane over the cuticular notch to round and thereby reduce the distention. E, Motion of the cupula is possibly the greatest near the crista (C) because of the wide subcupular space where the cupula is loosely attached. F, A contour head of the ciliary apparatus results in amplification of the axial displacement of the kinocilium as seen when comparing the paired models at 5 or 10". Even at 3", where the displacement is 3 pm, the change at the base of the kinocilium is significant.
P E R I P H E R A L V E S T I B U L A R R E C E P T O R S Y S T E M IN F R O G
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of central cupular displacement given by Oman and Young (1972) for a cupula which swings as a door so that displacement is in the shape of a wedge. On the other hand, their results correlate very closely to the cupular movement which would obtain if the volume displacement given by the authors were applied to an elastic diaphragm that has its greatest displacement near the crista at midpoint of its length. REFERENCES BREUER,J. (1891) Uber die Function der Otolithen-Apparate. Pfliigers Arch. ges. Physiol., 48, 195 306. DOHLMAN, G. F. (1969) The shape and function of the cupula. J. Laryng., 83, 43-53. DOHLMAN, G . F. (1971) The attachment of the cupulae, otolith and tectorial membranes to the sensory cell areas. Acta oto-luryng. (Stockh.), 71, 89-105. FLOCK, A. AND WERSALL,J. (1962) A study of the orientation of the sensory hairs of the receptor cells inthe lateral line organ of fish, with special reference to the function of the receptors. J. Cell Biol., 1 5 , 19-27. HILLMAN, D. E. (1969) New ultrastructural findings regarding a vestibular ciliary apparatus and its possible functional significance. Brain Res., 13, 407-412. HILLMAN, D. E. AND LEWIS, E. R. (1971) Morphological basis for a mechanical linkage in otolithic receptor transduction in the frog. Science, 174, 416-119. LOWENSTEIN, 0. AND WERSALL,J. (1959) A functional interpretation of the electronmicroscopic structure of the sensory hairs in the cristae of the elasmobranch Raja clavata in terms of directional sensitivity. Nature (Lond.), 184, 1807-1808. OMAN, C. M. AND YOUNG, L. R . (1972) Physiological range of pressure difference and cupula deflections in the human semicircular canal : theoretical considerations. In A. BRODAL AND 0. POMPEIANO (Eds.), Progress in Brain Research. Vol. 37. Basic aspects of central vestibular mechanisms. Elsevier, Amsterdam, pp. 529-539. SPOENDLIN, H. H. (1965) Ultrastructural studies of the labyrinth in squirrel monkeys. In The Role of the Vestibular Organs in the Exploration of Space. NASA SP-77, 7-22. STEINHAUSEN, W. (1934) Uber die direkte Beobachtung der Sinnes-Endstellen des inneren Ohres (mit Film). Zool. Anz., 36, 85-91. TRINCKER, D., (1962) The transformation of mechanical stimulus into nervous excitation by the labyrinthine receptors. Symp. SOC.exp. BioE., Biological Receptor Mechanisnis, 16, 289-316. WERSALL, J. (1956) Studies on the structure and innervation of the sensory epithelium of the cIistae ampullares in the guinea pig. Acra oto-laryng. (Srockh.), 126, 1-85. ZALIN,A. (1967) On the function of the kinocilia and stereocilia with special reference to the phenomenon of directional preponderance. J. Laryng., 81, 119-135.
Observations on Morphological Features and Mechanical Properties of the Peripheral Vestibular Receptor System in the Frog D. E. H I L L M A N
Division of Neurobiolofy, Department of Physiology and Biophysics, University of Iowa, Iowa City, l o wa, U S . A
.
Breuer (1891) noted that tuft-like structures projected from the surface of the receptor epithelium. He attributed the receptor function to the shearing motion of' these hairlike masses. With electron microscopy two types of cilia are found in the tuft (Wersall, 1956): (i) numerous stereocilia which are positioned over a relatively ridged cuticular plate that is placed at the luminal end of the cell and (ii) a single kinocilium which stands over a notch in this plate (Flock and Wersall, 1962; Spoendlin, 1965; Hillman, 1969) (Figs. 1, 2). Since all the kinocilia are on the same side of the tuft Lowenstein and Wersall (1959) were able to relate this morphological polarity to the activity within the vestibular nerve. A force which was directed toward the kinocilium caused an increase in the activity of the vestibular nerve while a decrease in activity was seen when the force was directed away from the kinociliary side of the cell. The action of the ciliary hairs in the otolithic organs following a positional change shows that the sterocilia have a sliding motion at their distal ends (Hillman and Lewis, 1971) (Fig. 3B-D). The kinocilia, however, are different in that the distal portion of the kinocilium, at least in the case of the frog, is attached to the adjacent stereocilia (Figs. 2, 3A). For this reason the kinocilium cannot slide at its distal end but is displaced at its base (Figs. 2,4). When a force is directed toward the kinociliary side of the cell, a plunging motion results on the kinocilium which produces a deformation of the cell membrane at the kinociliary base (Fig. 4A, B). At the same time a distension of the remaining membrane over the cuticular notch occurs as a protrusion of the cell in the notch region (Fig. 4A, B). When a force is applied in the opposite direction (toward the stereociliary side of the cell) the base of the kinocilium is raised while the stereocilia, which stand on the ridged cuticle, slide at their distal ends (Figs. 3B, 4C, D). This results in a rounding of the surface membrane of the cell over the notch region and thereby reduces the distension of the membrane at this active site (Fig. 4C, D). The result is a directional mechanism which is postulated to produce the conductive changes necessary for modulation of transmitter release and the activation of afferent fibers. The relationship between the ciliary apparatus and the otolithic membrane is compatible with the plunging motion of the kinocilium. In the case of the sacculus in References p . 75
70
D.E. H I L L M A N
Fig. 1 . Scanning electron micrograph showing the ciliary tufts on the saccular macula of the frog. The ciliary tuft is composed of stereocilia (S) and a single kinocilium (K) which has a bulb at its apex. The ciliary tufts are bent away from the kinociliary side of the cell. A filamentous mat (FB) which is over the supporting cells delineates the luminal surface of the receptor cells.
PERIPHERAL VESTIBULAR RECEPTOR SYSTEM IN FROG
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Fig. 2. Diagram to show the vestibular ciliary apparatus and its relationship to the apical end of the receptor cell in the frog. The stereocilia (S) stand on the cuticular plate (C) which is in the apical end of the receptor cell. The kinocilium (K) is positioned over a notch (N) in the cuticle where its base is in contact with the cell cytoplasm. Due to a filamentous attachment of the kinociliary bulb to adjacent stereocilia the pliable region in the area of the cuticular notch can yield to axial motion of the kinocilium. Two rows of stereocilia are cut at their base.
the frog, the bulb at the apex of the kinocilium (Figs. 1-3) is attached to the rim of a port in the otolithic membrane (Hillman and Lewis, 1971). In the utricle where the kinocilium is 3 to 4 times longer than the longest stereocilia the apical part of the kinocilium is embedded in the otolithic membrane. The otolithic membrane is attached to supporting cells by filament-like structures (Figs. 1, 3B-D) which cross the submembranous base to become part of the otolithic membrane (Hillman and Lewis, 1971). This submembranous zone is the shearing site for displacement between the supporting cells and the otolithic membranes (Hillman, in preparation). A displacement of the otolithic membrane draws the kinocilium and the adjacent stereocilia in the direction of its motion and thereby produces the axial motion in the kinociliary shaft which is related to the direction of the motion (Fig. 4). In the canal system the ciliary processes are 50 ,um or more in length. A row of stereocilia, 5 or 6, accompanies the kinocilium across the subcupular space where the kinocilium enters and is embedded in the cupula. Like the otolithic membrane, the subcupular space is crossed by fine filaments which anchor the cupuIa to the supporting cell (Dohlman, 1971). The action of the cupula is the subject of much controversy since varied opinions References p . 75
72
D. E. H I L L M A N
and observations exist (Steinhausen, 1934; Trincker, 1962; Zalin, 1967; Dohlman, 1969). It has been commonly accepted that the cupula acts as a swinging door with its axis on the crista. Numerous factors, however, indicate that such a movement is incompatible with the properties of such a structure. An alternative possibility is an elastic diaphragm which has a resiliency that would return it to the original position. The small displacement volume which Oman and Young (1972) calculated for physiological movement indicates that under the conditions of their study, the movement of the hairs would be negligible. An elastic diaphragm which is bound circumferentially to the ampullary wall, except for the crista where it is loosely bound by filaments which cross the wide subcupular space, would have its greatest displacement near the crista somewhat like the pendulum proposed by Zalin (1967) (Fig. 4E). This would result in the greatest amount of ciliary displacement for a given volume of endolymph. The cupular zone near the center of the crista would have minimal resistance and could allow for responses to small movements. On the other hand, during large endolymph changes (possibly non-physiological), attachment of a cupula to the crista through the kinocilia and filaments holds the cupular base to this structure while the apex breaks away (Hillman and Llinas, unpublished observation) and thereby gives the classical swinging door appearance seen by Steinhausen (1934), Dohlman (1969) and other investigators. To amplify the small movement which appears to be present during small angular deviations, the ciliary apparatus itself may bend in an arching manner which physically amplifies the axial motion of the kinocilium over and above that o f straight processes (Fig. 4F). This arching may be due in part to the pipe organ arrangement of the stereocilia which have a graded resistance so that bending away from the kinociliuni produces a pulley-like arch for the kinocilium (Fig. 3B). With motion toward the kinociliary side of the cell, the longer stereocilia have the least resistance so that bending occurs near the distal end of the kinocilium as a distal arch of the ciliary apparatus. Arching which results over a small length of the kinocilium can greatly enhance the displacement seen at the base of the kinocilium. In a model of a 50 pm long kino-stereociliary tuft which has a contoured bend the displacement at the base as significant with a 3 pm apical displacement (Fig. 4F). This niovement is in the realm
Fig. 3 . Transmission and scanning electron micrographs from the frog to show the relationship between the stereocilia and kinocilium due to displacement during fixation of the sacculus. A, Transmission electron micrograph showing the filaments which bind the apical end of the kinocilium (K) to an adjacent stereocilium (S). Small filamentous processes arise from a dense zone on the kinociliary membrane and traverse a cleft to join the membrane of the stereocilium. B, Scanning electron micrograph showing the ciliary tuft of the kinocilium (K) and numerous stereocilia which are bent away from the kinociliary side of the receptor cell (RC). A filamentous mat (FB) overlies the supporting cells. C , Scanning electron micrograph from the region of the striola of the saccular macula showing the opposed orientation of the ciliary tufts. D, A scanning electron micrograph showing two directionally opposed ciliary tufts which have been fixed while the gravitational force on the otolithic mass was changed by 180". The top stereociliary tuft is bent away from the kinociliary side of the cell while the lower tuft is bent toward the kinociliary side of the cell. The sliding between the stereocilia is obvious in this preparation. At the arrow the raised cell membrane over the cuticular notch is evident. B and D from Hillman and Lewis (1971).
P E R I P H E R A L V E S T I B U L A R R E C E P T O R SYSTEM I N F R O G
References p. 75
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D. E. H I L L M A N
A
C
E
Fig. 4. Diagrams and transmission electron micrographs to show the axial motion of the kinocilium due to directional displacement of the ciliary tuft. A and B, Motion toward the kinociliary side of the cell causes the kinocilium (K)to plunge into the cuticular notch (N) which results in a deformation and distention of the membrane in this region. C and D, Displacement away from the kinociliary side of the cell raises the base of the kinocilium causing the membrane over the cuticular notch to round and thereby reduce the distention. E, Motion of the cupula is possibly the greatest near the crista (C) because of the wide subcupular space where the cupula is loosely attached. F, A contour head of the ciliary apparatus results in amplification of the axial displacement of the kinocilium as seen when comparing the paired models at 5 or 10". Even at 3", where the displacement is 3 pm, the change at the base of the kinocilium is significant.
P E R I P H E R A L V E S T I B U L A R R E C E P T O R S Y S T E M IN F R O G
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of central cupular displacement given by Oman and Young (1972) for a cupula which swings as a door so that displacement is in the shape of a wedge. On the other hand, their results correlate very closely to the cupular movement which would obtain if the volume displacement given by the authors were applied to an elastic diaphragm that has its greatest displacement near the crista at midpoint of its length. REFERENCES BREUER,J. (1891) Uber die Function der Otolithen-Apparate. Pfliigers Arch. ges. Physiol., 48, 195 306. DOHLMAN, G. F. (1969) The shape and function of the cupula. J. Laryng., 83, 43-53. DOHLMAN, G . F. (1971) The attachment of the cupulae, otolith and tectorial membranes to the sensory cell areas. Acta oto-luryng. (Stockh.), 71, 89-105. FLOCK, A. AND WERSALL,J. (1962) A study of the orientation of the sensory hairs of the receptor cells inthe lateral line organ of fish, with special reference to the function of the receptors. J. Cell Biol., 1 5 , 19-27. HILLMAN, D. E. (1969) New ultrastructural findings regarding a vestibular ciliary apparatus and its possible functional significance. Brain Res., 13, 407-412. HILLMAN, D. E. AND LEWIS, E. R. (1971) Morphological basis for a mechanical linkage in otolithic receptor transduction in the frog. Science, 174, 416-119. LOWENSTEIN, 0. AND WERSALL,J. (1959) A functional interpretation of the electronmicroscopic structure of the sensory hairs in the cristae of the elasmobranch Raja clavata in terms of directional sensitivity. Nature (Lond.), 184, 1807-1808. OMAN, C. M. AND YOUNG, L. R . (1972) Physiological range of pressure difference and cupula deflections in the human semicircular canal : theoretical considerations. In A. BRODAL AND 0. POMPEIANO (Eds.), Progress in Brain Research. Vol. 37. Basic aspects of central vestibular mechanisms. Elsevier, Amsterdam, pp. 529-539. SPOENDLIN, H. H. (1965) Ultrastructural studies of the labyrinth in squirrel monkeys. In The Role of the Vestibular Organs in the Exploration of Space. NASA SP-77, 7-22. STEINHAUSEN, W. (1934) Uber die direkte Beobachtung der Sinnes-Endstellen des inneren Ohres (mit Film). Zool. Anz., 36, 85-91. TRINCKER, D., (1962) The transformation of mechanical stimulus into nervous excitation by the labyrinthine receptors. Symp. SOC.exp. BioE., Biological Receptor Mechanisnis, 16, 289-316. WERSALL, J. (1956) Studies on the structure and innervation of the sensory epithelium of the cIistae ampullares in the guinea pig. Acra oto-laryng. (Srockh.), 126, 1-85. ZALIN,A. (1967) On the function of the kinocilia and stereocilia with special reference to the phenomenon of directional preponderance. J. Laryng., 81, 119-135.