Motile responses of isolated guinea pig vestibular hair cells

231

Neuroscience Letters, 127 (1991) 231-236 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030443909100315V NSL 07828

Motile responses of isolated guinea pig vestibular hair cells Jean Valat, Corinne Griguer, Jacques Lehouelleur and Alain Sans INSERM U-254, Laboratoires de Neurophysiologie Sensorielle et de Neurophysiologie Cellulaire, U.S.T.L., Montpellier (France) (Received 5 February 1991; Revised version received 21 March 1991; Accepted 25 March 1991)

Key words: Isolated vestibular hair cell; Chemical stimulation; Mechanical stimulation; Motile response Vestibular hair cells were isolated from the guinea pig vestibule by a micromechanical non-enzymatic procedure. Perfusion with 125 mM K ÷ solution induced irreversible slow shortening of the necks in 42.8% of the hair ceils tested. Mechanical stimulation, creating a displacement of the hair bundle towards the kinocilium, induced either irreversible coiling or tilting of the neck of the cells, or reversible fast tilting of the cuticular plate (44.5% of tested cells). The response to the Ca 2 + antagonist, Flunarizine, suggested that these movements were calcium-dependent. We propose several explanations of the physiological role of these mechanisms and discuss the possibility that fast tilting of the cuticular plate is a physiological movement involving the hair cells at the periphery of the vestibular receptors. The regulation of the vestibular message at the apex of type I hair cells is also considered.

Morphological and electrophysiological studies carried out over the past ten years have clearly established the presence of active mechanisms in outer hair cells of the mammalian cochlea [1, 2, 10, 34, 35, 36]. Morphological studies have suggested the existence of a similar mechanism in vestibular hair cells [12, 23, 24, 27]. The main evidence is that cytoskeletal and contractile proteins have been detected at the apex of vestibular hair cells in mammals [6, 25, 29, 30]. Scarfone et al. [27] have also demonstrated the existence of synaptic microvesicles on the upper part of the nerve calyx, just below the level of cuticular plate. These hypothetical mechanisms could depend on calcium ions, which play a key role in transduction phenomena [15, 20-22]. We have investigated the motile properties of dissociated guinea pig vestibular hair cells using high-potassium and mechanical stimulation. The present results, some of which have been presented in preliminary reports [4, 11, 32], show that reversible tilting of the cuticular plate can be obtained by mechanical stimulation, while high potassium solutions induce irreversible shortening of the neck of the hair cells. Young guinea pigs (180-280 g) were anesthetized with ether and decapitated. The bullae were rapidly removed and the labyrinth opened. The cristae ampullaris were

Correspondence: J. Valat, INSERM U-254, Laboratoire de Neurophysiologie Sensorielle, U.S.T.L., 34095 Montpellier Cedex 05, France.

dissected out in 100 pl of fresh Hank's balanced salt solution (HBSS, osmolarity 285 mOsm); its composition (in mM) was: NaC1 137, KCI 5.4, KH2PO4 0.4, NazHPO4 0.4, MgSO4 0.8, CaC12 1.2, glucose 5.5; buffered with 5 mM HEPES to pH 7.4 and adjusted to 300 mOsm with 4 M NaC1 [7, 32]. The osmolarity was measured by determining the freezing point of the solution with an automatic osmometer (Roebling). The final NaC1 concentration was raised to 150 mM. Hair cells were isolated by mechanical dissociation in an experimental chamber on a glass coverslip previously treated with poly-L-lysine (0.2 mg/ml). The coverslip was inserted into the base of a Petri dish (Falcon, 1008) with the bottom partially removed to allow high power microscopic observation. The cells became attached to the glass after 10 min; the volume of the solution was then increased to 1 ml. All experiments were carried out at room temperature. Observations were made with an inverted microscope (Zeiss axiovert 35) using phase or interference contrast optics. The microscope was coupled to a video camera connected to a video monitor, and images were stored on a Sony U-Matic video cassette recorder. Chemical or mechanical stimulations were used. Chemical stimulations were performed by superfusion with modified HBSS containing 125 mM KCI and 17 mM NaCI (300 mOsm) for 10 min by means of a peristaltic pump. The cells were washed with normal HBSS solution for 15 min after treatment with the high potassium solution. Preliminary experiments were performed

232 to ensure that no morphological changes occurred in response to normal HBSS solution. The cells were also mechanically stimulated by deflecting the hair bundle (45 ° angle) with an angular acceleration of 7.103 rad .s-2. This stimulation was produced by impact excitation of a falling drop (1 /.tl) from a height of 2 mm above the surface of the bath. This ensured that the stimulus amplitude was always the same and it was easy to push the stereocilia in the direction of the kinocilium (efficient stimulus). The stimulated hair cells were carefully chosen so that the hair bundle of the cells was free and did not stick to the glass coverslip. The isolated hair cells appeared to conform to the histological criteria defined by Housley et al. [14], i.e. apparent opacity, smooth membrane surface and nongranular appearance. Control experiments were also performed to test the viability of the cells, using either a simultaneous double labeling procedure with fluorescein di-acetate and propidium iodide [16] or by measuring the resting potential of the cell placed in the whole-cell clamp configuration. The double labeling indicated that about 90% of the identified sensory cells could be maintened in vitro for 5 h after dissociation. The mean zero current resting membrane potential of cells placed in the whole-cell clamp configuration was - 5 1 . 1 + 11.6 mV (mean+S.D.; n = 5 3 ) throughout an experiment. This value is in agreement with those of frog [14] and guinea pig [37] vestibular hair cells and confirms that the isolated hair cells exhibited the characteristics of viable cells for several hours after cell isolation. Type I cells (Fig. I A) were particularly frequent in

preparations from cristae ampullaris. 37% of these cells had tilted cuticular plates and had hair bundle always bent in the direction opposite to the kinocilium (Fig. 1B). Chemical or mechanical stimulation of these 'tilted' cells evoked no motile response. Only hair cells with characteristics of type I hair cells (Fig. IA) were tested. Chemical stimulation. A total of 56 hair cells were stimulated with high K + (125 mM) solutions. 24 cells (42.8%) showed shortening of the neck region. The length changes were very slow, and after 20 min the typical type I hair cell shape had become spherical (Fig. 2). Perfusion with normal HBSS for several minutes following high K + stimulation not induce a return to the initial typical shape. There was a very discrete tilt of the hair bundle in some cells, with a shortening of the hair cell neck. A total of 32 cells (57.2%) showed no shape change under these experimental conditions. Controls (17 cells) showed no shape change when normal HBSS was perfused for 2 h. Mechanical stimulation. Eight of the 18 type I hair cells tested (44.5%) showed a motile response: Two hair cells fixed by their bases rolled up completely. The inclination of the hair bundle changed 30 ° over a period of 40 s. When the necks were wrapped around the soma, the cells looked like the tilted hair cells obtained by mechanical dissociation (Fig. ! B). Six hair cells attached to the support along their entire length, with their hair bundles remaining free, showed tilting of the stereocilia and the cuticular plate with a small (2/tm) downward movement of the neck. In 3 cases this tilting, which started immediately after the excitatory stimulation, was fast

Fig. 1. Dissociatedvestibulartype I sensoryhair cells. A: hair cell with a long straight neck. This type of hair cellsrepresented63%of total dissociated hair cells. B: hair cell with a curved appearance (37%of the dissociatedhair cell population), Interferentialdifferentialcontrast ( × 2500).

233

Fig. 2. Morphological changes of type I hair cell during a 10 min (between A and D) perfusion with 125 mM K ÷ Hanks solution. Notice the progressive shortening of the neck of the hair cell. This slow movement was not reversed by perfusion with normal solution (E and F). Phase contrast ( x 1100).

(less than 1 s) and irreversible. In the 3 other cases, the cells showed reversible movement when the stimulation displaced the hair bundle toward the kinocilium (Fig. 3A, B). Stimulation on the other side was without effect. There was a very clear contraction at the apex of the hair cell with a 45 ° tilt of the hair bundle and cuticular plate. This movement could be repeated several times (Fig. 3B). The tilting movement was relatively fast (5 s), and the hair bundle returned to a resting position after 7-12 s. This movement seemed to be calcium dependent; it did not occur after perfusion with HBSS containing the calcium antagonist flunarizine (10 -7 M) [13, 17], while the cuticular plate progressively relaxed (Fig. 3C). The presence of contractile proteins at the apex of vestibular hair cells suggests the existence of active mechanisms in the vestibule [6, 25, 30]. Several previous studies [5, 32, 37] have used dissociated mammalian vestibular epithelia to test this hypothesis in vitro. Chemical stimulation by high K ÷ solutions essentially induces modifications of cell length. This slow and irre-

versible change in the shape of the cells raises the possibility of osmolar effects of high K ÷ solution on the hair cells. Dulon et al. [7] showed that a change in the osmolarity of the external medium induces shortening of cochlear outer hair cells. Didier et al. [5] observed a tilt of the neck region of vestibular type I cells, accompanied by swelling of the cell bodies. The osmolarity did not change significantly during the time of the stimulation in our experiments. Nevertheless, it is difficult to believe that these movements could be physiological. There are several morphological and physiological arguments against such an effect: first, vestibular hair cells, unlike cochlea outer hair cells, are in close contact with afferent nerve fibers and supporting cells. Thus, vestibular hair cells cannot undergo large longitudinal movements as can the outer hair cells. Outer hair cells also have a special luminated cisternal system [8], which could explain their motility [3]; but vestibular hair cells do not appear to have such an organisation. Second, outer hair cell motility generates vertical forces on the tectorial mem-

234

brahe, while vestibular hair cells can act on the cupular membrane only in the tangential plane in the ampulla. Third, these movements are very slow, taking place over several minutes (> 20 min), which is too long for them to be involved in the control of transducing mechanisms.

i

The efficiency of vestibular hair cell mechanical stimulation depends on the rate of acceleration and the direction of the movement of the hair bundle with respect to the cuticular plate. This is probably why our positive resuits were obtained with a single mechanical stimulation

:! iii

Fig. 3. Reversible tilting of the cuticular plate and the hair bundle after mechanical stimulation. A I - A 4 and B1-B4: responses after two successive stimulations. The arrow indicates the direction of the stimulation (S), the arrow head the direction of response. C1-C4: stimulation in the presence of flunarizine (10-7 M) had no effect. Phase contrast ( × 2500).

235

which induced an excitatory displacement of the hair bundle. Two types of movement were identified: a coiling up of the neck around the soma and a reversible or irreversible tilting of the cuticular plate. These two types of movements could be correlated with the manner in which the cell adheres to the support. The tilting movements were observed in the cells fixed along their entire lengths, whereas the coiling up movements were exhibited by the cells fixed only by their bases. These latter cells, free of any constraint, could express all of their motile potential. The fast tilting of the cuticular plate and hair bundle induced by mechanical stimulation are probably closer to physiological responses than the slow movement induced by chemical stimulation. Several morphological results favour this proposition. There is an actin ring surrounding the cuticular plate and myosin and tropomyosin are present at the apex of the cell [25]. The rigidity of the cuticular plate may be dependent, via a calciumregulated mechanism, on a spectrin-related protein (fodrin) which cross-links actin filaments and anchors the cuticular plate to the apical cell membrane [27]. Synaptic vesicles have been found in the apical portion of the nerve calyx which completely encloses type I hair cells: this calyx might be involved in short-loop feedback control of the apex of type I hair cells [28]. Finally, protrusions of the luminal surface of vestibular hair cells into the endolymphatic fluid have been observed in freezefracture and transmission electron microscope studies

[9]. Nevertheless, if the apex of the hair cells undergo active fast movements in the vestibular organs in vivo, then such movements might be expected to involve only some of the hair cells. Scanning electron microscope investigations of the mouse cristae indicated that the hair cells situated at the top of the cristae have short, free standing stereocilia, while the cells at the edge and at the bottom had tall kinocilia and stereocilia inserted in the cupula [19]. In the utricule and saccule, the hair bundles of type I hair cells at the peripheral and parastriola regions are inserted into a chamber of the honeycomb, while the cilia are free standing in the striola region [18]. The motile responses of the hair cells in the vestibule of mammals may have two possible explanations. First, by comparison with the cochlea, the hair cells situated at the periphery of the vestibular organs could act as the outer hair cells and pull on the cupula or the otoconial membrane, with fast tilting of their cuticular plate and the stereocilia, thus modulating the vestibular message of the more centrally situated cells. Second, these movements, which are probably Ca2÷ dependent, may be implicated in control of the transduction of the vestibular message [26, 28, 34].

The authors are grateful to J. Boyer for editorial assistance. This work was supported by grant 88-135 from the Direction des Recherches Etudes et Techniques (D.R.E.T.). 1 Brownell, W.E., Microscopic observations of cochlear hair cells motility, Scanning Microsc., III (1984) 1401-1406. 2 Brownell, W.E., Bader, C.R., Bertrand, D. and De Ribeaupierre, Y., Evoked mechanical response of isolated cochlear outer hair cells, Science, 227 (1985) 194-196. 3 Brownell, W.E., Outer hair cell electromotility and otoacoustic emissions, Ear and Hearing, I 1 (1990) 82-92. 4 Devau, G., Valat, J. and Sans, A., Mise en 6vidence de m6canismes actifs dans les cellules sensorielles vestibulaires isol6es de cobaye, 36me coll. intern. Neurosci., Montpellier, France, 1989, Abstr. B66. 5 Didier, A., Decory, L. and Cazals, Y., Evidence for potassium induced motility in type I vestibular hair cells in the guinea pig, Hear. Res., 46 (1990) 171-176. 6 Drenckhahn, DI, Sch/ifer, T. and Prinz, M., Actin, Myosin and associated proteins in the vertebrate auditory and vestibular organs: immunocytochemical and biochemical studies. In D.G. Drescher (Ed.), Auditory Biochemistry, C.C. Thomas, Springfield, IL, 1985, pp. 317 335. 7 Dulon, D., Aran, J.M. and Schacht, J., Potassium-depolarization induces motility in outer hair cells by an osmotic mechanism, Hear. Res., 32 (1988) 123-129. 8 Evans, B.N., In vitro correlates of outer hair cell vulnerability: electromechanical and ultrastructural observations, Hear. Res., in press. 9 Favre, D., Bagger-Sj6back, D., MBiene, J.P. and Sans, A., Freezefracture study of the vestibular hair cell surface during development, Anat. Embryol., 175 (1986) 69-76. 10 Flock, A., Flock, B. and Ulfendahl, M., Mechanisms of movement in outer hair cells and a possible structural basis, Arch. Otolaryngol., 243 (1986) 8340. 11 Griguer, C., Chabbert, C., Devau, G., Valat, J., Sans, A. and Lehouelleur, J., Are calcium ions implicated in motile responses of mammalian vestibular hair cells, 27th I.E.B. Meet., Stockholm, 1990, Abstr. 100. 12 Hirokawa, N. and Tilney, L.G., Interactions between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear, J. Cell Biol., 95 (1982) 249-2. 13 Holmes, B., Brodgen, R.N., Heel, R.C., Speight, T.M. and Avery, G.S., Flunarizine. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use, Drugs, 27 (1984) 6-44. 14 Housley, G.D., Norris, C.H. and Guth, P.S., Electrophysiological properties and morphology of hair cells isolated from the semicircular canal of the frog, Hear. Res., 38 (1989) 259-276. 15 Hudspeth, A.J. and Lewis, R.S., Kinetic analyses of voltage and ion-dependent conductances in saccular hair cells of the bull-frog Rana catesbeiana, J. Physiol., 400 (1988) 237-274. 16 Jones, K.H, and Senft, J.A., An improved method to determine cell viability by simultaneous staining with fluorescein diacetatepropidium iodide, J. Histochem. Cytochem., 33 (1985) 77-79. 17 Kubo, K., Matsuda, Y., Kase, H. and Yamada, K., Inhibition of calmodulin-dependent cyclic nucleotide phosphodiesterase by flunarizine a calcium-entry blocker, Biochem. Biophys. Res. Commun., 124 (1984) 315-321. 18 Lim, D.J., Fine morphology of the otoconial membrane and its relationship to the sensory epithelium, Scanning Electron Microsc., III (1979) 929 938.

236 19 MBiene, J.P. and Sans, A., Differentiation and maturation of the sensory hair bundles in the fetal and postnatal vestibular receptors of the mouse: a scanning electron microscopy study, J. Comp. Neurol., 254 (1986) 271-278. 20 Ohmori, H., Studies of ionic currents in the isolated vestibular hair cell of the chick, J. Physiol., 350 (1984) 561-581. 21 Ohmori, H., Mechano-electrical transduction currents in isolated vestibular hair cells in the chick, J. Physiol., 359 (1985) 189-217. 22 Ohmori, H., Mechanical stimulation and Fura-2 fluorescence in the hair bundle of dissociated hair cells of the chick, J. PhysioL., 399 (1988) 115 137. 23 Ross, M.D. and Bourne, C., Interrelated striated elements in vestibular hair cells of the rat, Science, 220 (1983) 622-624. 24 Sans, A., Ultrastructural study of striated organelles in vestibular sensory cells of human fetuses, Anat. Embryol., 179 (1989) 457 463. 25 Sans, A., Atger, P., Cavadore, C. and Cavadore, J.C., Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats, Hear. Res., 40 (1989) 117 125. 26 Sans, A., Etchecopar, B., Brehier, A. and Thomasset, M., Immunocytochemical detection of vitamin D-dependent calcium binding protein (CaBP-28k) in vestibular sensory hair cells and vestibular ganglion neurones of the cat, Brain Res., 364 (1986) 19(~ 194. 27 Scarfone, E., Dem6mes, D., Perrin, D., Aunis, D. and Sans, A., ~t-Fodrin (brain spectrin) immunocytochemical localization in rat vestibular hair cells, Neurosci. Lett., 93 (1988) 13-18. 28 Scarfone, E., Dem6mes, D., Jahn, R., De Camilli, P. and Sans, A., Secretory function of the vestibular nerve calix suggested by presence of vesicles, synapsin I, and synaptophysin, J. Neurosci., 8 (1988b) 4640-4645.

29 Slepecky, N. and Chamberlain, S.C., Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells, Hear. Res., 20 (1985) 245-260. 30 Sobin, A. and Flock, A., Immunohistochemical identification and localization of actin and fibrin in vestibular hair cells in the normal guinea pig and in a strain of the waltzing guinea pig, Acta Otolaryngol., 96 (1983) 407--412. 31 Valat, J., Devau, G., Dulon, D. and Sans, A., Vestibular hair cells isolated from guinea pig labyrinth, Hear. Res., 40 (1989a) 255-260. 32 Valat, J., Devau, G., Griguer, C., Zidanic, M., Lehouelleur, J. and Sans, A., Vestibular hair cells isolated from guinea pig labyrinth, 26th I.E.B. Meet., Paris, France, 1989b, Abstr. P72. 33 Yamashita, M. and Ohmori, H., Synaptic responses to mechanical stimulation in calyceal and bouton type vestibular afferents studies in an isolated preparation of semicircular canal ampullae of chicken, Exp. Brain Res., 80 (1990) 475488. 34 Zenner, H.P., Motile response in outer hair cells, Hear. Res., 22 (1986) 1 14. 35 Zenner, H.P., Motility of outer hair cells as an active actinmediated process, Acta Otolaryngol., 105 (1988) 39-44. 36 Zenner, H.P., Zimmermann, V. and Schmitt, U., Reversible contraction of isolated mammalian cochlear hair cells, Hear. Res., 18 (1985) 127 133. 37 Zenner, H.P., Zimmermann, U. and Gitter, A.H., Cell potential and motility of isolated mammalian vestibular sensory cells, Hear. Res., 50 (1990) 289-294. 38 Zimmermann, V., Reuter, G., Shi Hong and Zenner, H.P., The motility of isolated mammalian vestibular sensory cells. Proc., 18th G6ttingen Neurobiol. Conf., Brain-Perception-Cognition, 1990, Abstr. 171.