During development of the guinea pig cochlea, motile activity of outer hair cells has been found to coin- cide with the presence of the subsurface cisternae (Pujol ...
Journal of Cell Science 104, 1137-1143 (1993) Printed in Great Britain © The Company of Biologists Limited 1993
1137
Dissociation between the calcium-induced and voltage-driven motility in cochlear outer hair cells from the waltzing guinea pig Barbara Canlon and Didier Dulon Department of Physiology II, Karolinska Institute, S-104 01 Stockholm, Sweden and Laboratoire d’Audiologie Experimentale, INSERM Unit 229, Hôpital Pellegrin, 33076 Bordeaux, France
SUMMARY The waltzing guinea pig, possessing an hereditary progressive deafness, shows pathology to the actin-bearing structures within the hair cells of the organ of Corti. In particular, the affected structures include the stereocilia, the cuticular plate and, as shown in the present study, swollen and disorganized subsurface cisternae. To test whether this pathology affected outer hair cell motility, cells were isolated from waltzing guinea pigs and their age-matched controls and were subjected to either electrical or chemical stimulation. Visual detection thresholds and the magnitude of the electricallyinduced length changes were equivalent for both groups. However, when intracellular calcium was increased with either the calcium ionophore, ionomycin or Ca2+/ATP (under permeabilized conditions with DMSO), length changes were significantly reduced for the outer hair cells from waltzing guinea pigs compared to the controls. The average percent length increase induced by 10 µM ionomycin for the outer hair cells from control animals was 2.3 ± 1.7 whereas for postnatal day 4 waltzing guinea pigs it was 1.3 ± 1.7. Postnatal day 7 and 10 waltzing guinea pigs responded with significantly smaller percent length changes. The intracellular con-
centration of ionic calcium increased similarly for both groups after the application of ionomycin as revealed with the indicator fluo-3. In the permeabilized cells in the presence of Ca2+/ATP, control cells responded with a percent length change of 3.5, whereas, age-matched waltzing outer hair cells responded with barely detectable length changes. When the osmolarity of the external medium was reduced, both the control and waltzing outer hair cells responded with a length change that was proportional to the change in osmolarity, indicating the capability of passive length changes. In summary, the voltage-dependent motility of isolated outer hair cells from the waltzing guinea pigs is intact, whilst the slow calcium-dependent motility is abnormal. As a result of these findings, the outer hair cells from the waltzing guinea pig allow separations between the slow, metabolically dependent length changes induced by chemical stimulation and the fast, metabolically independent length changes induced by electrical stimulation.
INTRODUCTION
cise subcellular structure within the outer hair cell, responsible for each type of motility, has been determined. One hypothesis is that the subsurface cisternae, along the plasma membrane (Saito, 1983), are involved in the slower type of force generation (Flock et al., 1986). Such a mechanism is believed to include the release of calcium from the cisternae, analogous to the sarcoplasmic reticulum found in muscle. During development of the guinea pig cochlea, motile activity of outer hair cells has been found to coincide with the presence of the subsurface cisternae (Pujol et al., 1991). Alternatively, a voltage-sensitive force generator that lies in the plasma membrane has been suggested to underlie the rapid outer hair cell length changes (Kalinec et al., 1992). A third possible structure includes the stereocilia, the stiff rods that are anchored into the cuticular plate. The stereocilia bundle detects, with a high degree of sensitivity, mechanical forces arising from acoustic stimulation (Crawford and Fettiplace, 1985). Force generation by
Active mechanical processes are evident in the auditory peripheral organ, the cochlea. Since the outer hair cells can change their length when stimulated electrically, chemically or mechanically, and generate acoustic emissions from the cochlea (Mountain, 1980), they are believed to be the structural entity responsible for the active mechanism. The outer hair cells are capable of undergoing relatively slow, metabolically dependent, length changes induced by chemical (Zenner, 1986; Dulon et al., 1990) or mechanical (Canlon et al., 1988, 1991) stimulation. This type of motile response is believed to involve contractile proteins. On the other hand, outer hair cells can also respond to electrical stimulation. These fast, ATP-independent length changes are believed to operate at acoustic frequencies (Brownell, 1984; Ashmore, 1987) and seem not to depend on actomyosin interactions. Neither the underlying mechanism nor the pre-
Key words: actin defect, calcium, cochlea, hearing loss, motility, outer hair cells
1138 B. Canlon and D. Dulon the stereocilia, however, has been found only in non-mammalian auditory systems. In this study we investigated the response of isolated outer hair cells from the waltzing guinea pig to chemical and electrical stimulation. The waltzing guinea pig is a strain of pigmented guinea pig that has a genetically induced progressive deafness (Ernstson, 1970). An elevated hearing threshold is evident at birth and rapidly progresses over the first month of life (Wit and Nijdam, 1984; Canlon et al., unpublished observations). In conjunction with the progressive loss of auditory sensitivity, a systematic degeneration of the hair cells from the organ of Corti occurs. The pattern of degeneration is such that the third row outer hair cells are the first to show pathology followed by the second row, then the first row, and finally the inner hair cells are affected (Ernstson, 1971; Canlon et al., unpublished observations). The prominent morphological alterations affect those cellular structures containing high concentrations of actin. These structures include the stereocilia and the cuticular plate. In addition, the present study shows abnormal subsurface cisternae along the plasma membrane of outer hair cells from the waltzing guinea pigs. This study was initiated on the basis of these morphological findings. The question was raised as to whether or not an outer hair cell possessing an altered actin distribution would be capable of length changes induced by either chemical or electrical stimulation. In order to address this question outer hair cells were isolated from waltzing guinea pigs at different postnatal ages and their motile capacity investigated. MATERIALS AND METHODS Animals The waltzing guinea pigs are maintained in our own animal colony. A normal adult female guinea pig is mated with a waltzing adult male guinea pig and the litter consists of both normal and waltzing guinea pigs in a ratio of 1:1. The most characteristic behavior of the waltzing guinea pig is its circling gait and lack of a nystagmus when rotated. There are no differences between the controls and the waltzing guinea pigs with respect to either the body weight at birth or during postnatal development. The age of the waltzing guinea pigs used in this study ranged from postnatal day 4 through day 20. A mild hearing loss is found already by day 4, which progresses to a moderate hearing loss of approximately 35 dB by day 20.
Electron microscope techniques For scanning electron microscopy, the cochleae were removed and fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4. The perilymphatic space was opened at the oval and round windows and at the apex. The fixative was gently circulated through the cochleae. After two hours, the cochleae were postfixed in 1% OsO4 in 0.1 M sodium cacodylate buffer. The cochleae were then washed in buffer with progressively lower concentrations until the final changes were pure water. After microdissection and removal of the tectorial membrane the specimen was immersed in saturated, filtered, freshly made thiocarbohydrazide for 15 minutes. Following washes in distilled water, the cochleae were immersed in 2% OsO4 for 2 hours, rinsed, and placed in 1% tannic acid for 10 minutes, then transferred to 2% tannic acid for at least 4 hours. After the final washing, the specimens were dehydrated in a graded series of ethanol up to 70%, then placed in 70% acetone and fur-
ther dehydrated in increasing concentrations of acetone, up to 100%. They were then dried in a critical point dryer in CO2, after which they were coated with gold in a sputter and examined in a Philips 505 scanning electron microscope. For transmission electron microscopy, the bullae were removed and placed in 2% glutaraldehyde in 0.1 M Sorensons buffer with 0.2% tannic acid for 2 hours. The cochleae were then rinsed with buffer and post-fixed with 1% OsO4 in buffer for 1 hour. The cochleae were dehydrated in a graded ethanol series and infiltrated with epoxy resin (Agar). After polymerization, the cochleae were cleaved axially and the area around the 15 mm distance from the round window cut out and remounted on blocks that were thin sectioned and stained with uranyl acetate and lead citrate. Sections were placed on Formvar coated grids and examined by transmission electron microscopy with a Zeiss EM 109 microscope and photographed.
Isolation of outer hair cells Guinea pigs were decapitated and the temporal bones removed, placed in culture medium, pH 7.4 at 300 mosmol/ml (Leibovitz L15; GIBCO), and the bony walls of the cochleae were opened. The concentrations of the major ion constituents of the medium in mM are: Na +, 135; K+, 5.8; Ca2+, 1.26 and Cl−, 147. All experiments were carried out at room temperature. The desired region of the organ of Corti was dissected free and mechanically separated by suction into a pipette with a constricted tip. Cells from the midto apical portion of the cochlea corresponding to the 13 to 18 mm distance from the round window were selected and placed in a 200 µl chamber containing L-15 culture medium. The osmolarity was checked before, during and at the end of the experiment using an osmometer (Roebling, Germany) which employs the freezing point depression technique. The osmolarity was maintained by the addition of distilled water to the chamber at regular intervals. The cells were viewed in either differential interference contrast optics, or epi-fluorescence in a Zeiss Axiovert 35 inverted microscope equipped with a Hamamatsu C3077 video camera with a contrast enhancement unit (Hamamatsu C2400). The maximal magnification on the TV monitor was ×2,500. Outer hair cells exhibiting pathological signs such as cytoplasmic granulations, Brownian motion, a displaced nucleus, blebs protruding from the cuticular plate, or any other pathological signs were not used for investigation. At the end of each experiment the osmolarity was checked, and only when the value was within normal limits were the results accepted. Experiments never exceeded 3 hours after the isolation of the outer hair cells. Cell images were digitalized on-line and the image stored for subsequent analysis of length using an Image Pro II image processor (Data Translation).
An objective test for cell viability Outer hair cell viability was determined according to the method of Dulon et al. (1989). Cells were simultaneously double stained by fluorescein diacetate (FDA) and propidium iodide (PI). Stock solutions of 5 mg FDA (Sigma) in 1 ml acetone and 350 µg of PI (Sigma) in 1 ml of L-15 culture medium were prepared. Mixed FDA and PI working solutions were freshly prepared in L-15 culture medium at concentrations of 10 µg FDA and 5 µg PI/ml. The cells were stained for 3 minutes and then rinsed in L-15. The percentage of viable/non-viable cells was determined at different times by counting the cells using the appropriate wavelength for the excitation of the fluorescently labelled compounds. FDA easily passes through cell membranes and is hydrolyzed by esterases to produce fluorescein. As a consequence, fluorescein accumulates inside viable cells and emits a green fluorescence when excited with blue light (450-490 nm). PI, on the other hand, is excluded by cells with intact membranes and stains the nuclei of nonviable cells red when excited by green light (515-560 nm).
Waltzing guinea pig outer hair cells 1139 Chemical stimulation Calcium-dependent motility Isolated outer hair cells were exposed to a final concentration of 10 µM ionomycin (Sigma) dissolved in DMSO (0.1%). Prior to and 3 minutes post application of 10 µM ionomycin, or DMSO, outer hair cell images were digitalized on-line and the image stored for subsequent analysis of length using an Image Pro II image processor (Data Translation). It has been previously demonstrated that 10 µM ionomycin causes an increase in intracellular calcium in outer hair cells (Dulon et al., 1990). Control cells were incubated in 0.1% DMSO. For the permeabilization of the plasma membrane to Ca2+ and ATP, isolated outer hair cells were treated with 1% DMSO for 1 minute, rinsed, then exposed to CaCl2 (1.25 mM) and ATP (0.1 mM; Sigma) for 3 minutes. The addition of ATP is required because in permeabilized cells the ATP is lost, in contrast to the intact cells where internal ATP is maintained. ATP has been shown to be a necessary requirement for actin-dependent motility of outer hair cells (Zenner, 1986). Cell images were digitalized on-line and the image stored for subsequent analysis of length as described above. Solutions for control cells were without calcium and with 1 mM EGTA (Sigma).
Calcium-independent motility Changes in osmolarity were achieved by adding a defined amount of distilled water to the chamber. Shape changes were monitored at 3 minutes post superfusion and the images were stored for future analysis.
any movement of the cell body or the stereocilia. The detection threshold was determined by either increasing the stimulus from below threshold until movement could just be detected, or by starting above an estimated threshold and decreasing stimulus intensity until threshold movement was observed. The intensity of the signal was determined directly from the oscilloscope (Tektronix 475). The response of the cells to electrical stimulation was recorded on video tape (Panasonic S-VHS, model FS1000) and later analyzed for the magnitude of length changes that occurred during stimulation.
RESULTS Morphology Fig. 1 shows two scanning electron micrographs (A and B) of the organ of Corti from a postnatal day 15 (PN 15) control guinea pig and a waltzing guinea pig (also PN 15), respectively. The control organ of Corti shows the typical well ordered array of the three rows of outer hair cells and the characteristic V-form of their stereocilia. In contrast, the organ of Corti from the 15 day old waltzing guinea pig shows abnormal stereocilia and cuticular plates. The stere-
Monitoring of the intracellular calcium concentration The calcium indicator fluo-3 and confocal laser scanning microscopy were used to monitor changes in [Ca2+]i during ionomycin stimulation in normal and waltzing guinea pig outer hair cells. The isolated hair cells were loaded with the fluorescent probe fluo-3 by an exposure to 2 µM fluo-3/AM at room temperature for 30 min. Optical fluorescent slices of the cells were obtained at video rates with a fast laser scanning confocal microscope (Odyssey, Noran Instruments, Inc., Middleton, WI, USA). This system makes use of an acousto-optic deflector and a galvanometric mirror for a complete x,y laser scan at 25 frames/s with 512/480 pixels resolution. The confocal system was attached to a Leitz Fluovert inverted microscope and was equipped with an argon ion laser and a fluorescein filter cartridge. The laser beam was focused by a 60× objective lens (Olympus Plan-Apo, numerical aperture of 1.3) and fluorescence was reflected by a dichroic mirror, through a 25 µm slit to the photomultiplier, allowing optical sections of approximately 0.5 µm. The video detector output was recorded on a tape recorder (U-Matic, Sony) and subsequently analyzed with a CompuAdd 386SX host computer using the image processing and analysis system Image-1 (Universal Imaging Corporation, West Chester, Penn, USA).
Electrical stimulation Extracellular Ag-AgCl electrodes, insulated except at the tip, and separated by a distance of about 200 µm were used for passing alternating current to isolated outer hair cells. The electrode pair was lowered into the culture medium containing the outer hair cells and oriented in relation to the basal pole of individual outer hair cells by rotating the microscope stage (Kachar et al., 1986). The stimulus consisted of a 10 Hz alternating sinusoidal signal (Signal generator J3, Advance Instruments) with a variable control for intensity. The voltage waveform was displayed on an oscilloscope screen. A visual detection threshold of outer hair cell movement was obtained for each cell. Movement was defined as
Fig. 1. Scanning electron micrograph of the organ of Corti from a PN 15 control (A) and a PN 15 waltzing guinea pig (B) from the 16 mm distance from the round window. The surface of the control organ of Corti shows an orderly array of hair cells and their stereocilia. The organ of Corti from the waltzing guinea pig frequently shows giant stereocilia and a bulging apical surface of the hair cell. Bar, 10 µm.
1140 B. Canlon and D. Dulon ocilia can increase their length by a factor of 2. The outer hair cells, showing giant and fused stereocilia, frequently have a bulging apical cell surface. Fig. 2A and B shows transmission electron micrographs of the plasma membrane of an outer hair cell from a postnatal day 7 (PN 7) control (A) and a PN 7 waltzing guinea pig (B). Both outer hair cells were obtained from the 15 mm distance from the round window. The outer hair cell from the control guinea pig shows the expected orderly array of the subsurface cisternae, with mitochondria in close proximity to the cisternae. The cisternae consist of multilayers along the cytoplasmic face of the plasma membrane. The waltzing guinea pig, on the other hand, demonstrates disorganized and swollen cisternae. The mitochondria in the outer hair cells of the waltzing guinea pig are more often found in the central portion of the cytoplasm rather than along the subsurface cisternae. The appearance of the mitochondria is often different from the control cells such that they are often twice as long and more slender. It is believed that the alterations to the subsurface cisternae are not a result of fixation artefacts, since other intracellular structures appear indistinguishable from the control material. Outer hair cell motility Viability An objective test for viability was employed to determine if the outer hair cells from the waltzing guinea pig could tolerate the isolation procedure. A total of 368 outer hair cells isolated from 3 control animals (PN 7) and a total of 166 outer hair cells isolated from 2 waltzing guinea pigs (PN 7) were studied. The viability of all the cells in the chamber was determined by counting all the ‘green’ outer hair cells and all the ‘red’ outer hair cell nuclei that were
present. The viability of the cells was tested at various times after isolation for up to three hours (Fig. 3). Both groups followed the same time course for degeneration over a three-hour period and therefore it is concluded that the outer hair cells from the waltzing guinea pig can tolerate the isolation procedure. This finding is essential for the interpretation of the chemically and electrically induced length changes of the outer hair cells. Chemical stimulation Calcium-dependent motility Calcium-induced outer hair cell motility has been observed in isolated outer hair cells under two different conditions: (1) in intact cells treated with ionomycin, elongation, through circumferential contraction, resulted in reductions in cell diameter (Dulon et al., 1990) and (2) permeabilization of the plasma membrane to calcium and ATP with detergents produced shortening of the cells (Zenner, 1986; Flock et al., 1986). These results indicate that the contractile apparatus behaves differently in permeabilized cells, compared to intact cells, presumably due to the dissolving action of the detergent on the plasma membrane (Dulon et al., 1990). In this study, both types of calcium stimulation were monitored in outer hair cells: (a) in intact cells with the ionophore, ionomycin and (b) in permeabilized cells with calcium and ATP. The effect of 10 µM ionomycin was studied on a total of 42 control and 44 waltzing guinea pig outer hair cells (Table 1). As previously reported (Dulon et al., 1990), outer hair cells respond to ionomycin with an increase in calcium and a consequent increase in length. Of the 34 cells obtained from 3 control animals at postnatal day 7, a mean percentage length increase of 2.7 was observed. The outer hair
Fig. 2. Transmission electron micrograph of a longitudinal section showing the plasma membrane in the supranuclear region of an outer hair cell, obtained from the 15 mm distance from the round window. (A) Control (PN 7), showing the subsurface cisternae lining the plasma membrane in an orderly manner with mitochondria in close proximity. (B) Waltzing guinea pig (PN 7) showing that the subsurface cisternae are swollen and disorganized. Bar, 0.5 µm.
Waltzing guinea pig outer hair cells 1141 Table 1. Effect of ionomycin on outer hair cell length Animal w− w− w+ w+ w+
PN7 PN10 PN4 PN7 PN10
% Length change
n
2.7 ± 1.9 1.9 ± 1.5 1.3 ± 1.7 0.3 ± 0.3 0.1 ± 0.8
34 8 18 13 13
w+ w−
Fig. 3. The percentage of viable/non-viable outer hair cells was determined as described in the Materials and Methods. The outer hair cells from the control group (open circles, w+) and waltzing guinea pigs (closed circles, w−) are represented.
cells (n=8) from one animal at postnatal day 10 showed a mean percent length increase of 1.9. The difference in these two control animals was not significantly different. A total of 16% of the control cells were unresponsive to ionomycin. Isolated outer hair cells from 2 waltzing guinea pigs at postnatal day 4 (n=18) also responded with a length increase when exposed to ionomycin, yet the magnitude of the length change was significantly less than for the control cells. The mean and standard deviation of the percent length change of these cells was 1.3 ± 1.7. This difference in the outer hair cell length change between control and waltzing guinea pigs is statistically significant (Student’s ttest, P < 0.01). A total of 7, or 38%, were unresponsive to ionomycin. Increasing the concentration of ionomycin (2×) for these unresponsive cells was not a successful means of producing a length change. When outer hair cells from 2 waltzing guinea pigs (PN 7) were studied (n=13), 38% were unresponsive and those that increased their length did so minimally. The mean percent length change was 0.3 ± 0.3. The outer hair cells (n=13) from 3 waltzing guinea pigs at postnatal day 10 also responded to ionomycin with a min-
imal increase in length (0.3 ± 0.5 s.d.); however, sixty-one percent of these outer hair cells did not respond at all. The length changes observed for the postnatal day 7 and 10 waltzing guinea pigs were significantly different from the control values (Student’s t-test, P < 0.001). In order to determine if the solute for ionomycin, DMSO, could induce length changes, outer hair cells (n=14) from 3 control guinea pigs were incubated in 0.1% DMSO for three minutes. This concentration corresponds to the final concentration added with the ionomycin to the chamber. The cells exposed only to the 0.1% solution of DMSO did not increase their length and maintained a healthy appearance. Confocal imaging using the fluorescent calcium-sensitive probe, fluo-3, was used to verify if ionomycin was able to increase the intracellular concentration of free calcium in the mutant hair cells as previously described in normal cells. The results indicate that the largely reduced mechanical responses of the waltzing guinea pig hair cells was not due to the lack of increase in [Ca2+]i during ionomycin stimulation. The confocal image analysis of the Ca2+ response in one of these cells is shown in Fig. 4. All the 9 outer hair cells observed increased their fluo-3 fluorescence level upon ionomycin stimulation as previously described for normal outer hair cells (Dulon et al., 1990), indicating that ionomycin had a similar ionophoric action for Ca2+ in both the waltzing guinea pigs and their age-matched controls. After the cells were permeabilized with DMSO and exposed to 1.25 mM CaCl2 and 0.1 mM ATP, outer hair cells from 3 control animals (PN 7) responded by a reduc-
Fig. 4. (A) confocal laser imaging of Ca2+ with fluo-3 before (left) and after (right) the addition of 10 µM ionomycin to the outer hair cell from a waltzing guinea pig. (B) The calcium kinetics for the outer hair cell. The arrow indicates the addition of ionomycin.
1142 B. Canlon and D. Dulon Table 2. Visual detection thresholds for outer hair cell length changes induced by electrical stimulation Animal w− w− w+ w+ w+
PN7 PN20 PN7 PN10 PN20
Threshold (V)
n
1.0 ± 0.3 1.1 ± 0.6 1.2 ± 0.3 1.2 ± 0.5 1.3 ± 0.3
26 20 10 19 40
tion in cell length (n=11). The mean value and standard deviation of the percent length change was 3.5 ± 1.9. Of the 11 cells studied, only one cell did not respond to the Ca2+/ATP. In contrast, the mean percent length change of outer hair cells (n=15) from 3 waltzing guinea pigs (PN 7) studied was 0.7 ± 0.3. Of the 15 cells that were studied, 10 cells did not respond with a length change (66.6%). The difference in outer hair cell length changes induced by Ca2+/ATP between control and waltzing guinea pigs was statistically significant (Student’s t-test, P < 0.001). In the presence of 1 mM EGTA and the absence of CaCl2, length changes of outer hair cells from 3 control animals (PN 7; n=10) were not observed. The results from the calcium-induced motility demonstrate a concomitant loss of responsiveness for the two different methods used to study calcium motility in the outer hair cells from the waltzing guinea pig, indicating that they are based on a similar contractile process. Osmotic stimulation When the osmolarity of the external medium was reduced either by 4% or 8%, the outer hair cells from 2 control animals (PN 7) responded with a decrease in length. During hypo-osmotic conditions the cell decreases in length due to water influx. The length change was proportional to the change in osmolarity in a similar fashion to previous reports (Dulon et al., 1988). A reduction in osmolarity by 4% gave rise to a mean percent length change of 3.8 ± 1.2 (n=9), and a change in osmolarity by 8% gave a mean percent length change of 7.3 ± 1.0 (n=5). Outer hair cells from 2 waltzing guinea pigs (PN 7) responded to a 4% reduction in osmolarity, with a mean percent length change of 3.9 ± 1.0 (n=7) and with an 8% reduction in osmolarity the mean percent length change was 8.4 ± 0.9 (n=5). All of the outer hair cells from the control guinea pigs, as well as from the waltzing guinea pigs responded to the changes in osmolarity. No significant differences in the outer hair cell length change, induced by decreases in osmolarity, were observed between control and waltzing guinea pigs (Student’s t-test, P < 0.3). The results indicate that the outer hair cells from the waltzing guinea pigs have the capacity to undergo passive length changes. Electrical stimulation A grand total of 115 outer hair cells were tested for a motile response to electrical stimulation (Table 2). Forty-six control outer hair cells (n=26) from 2 postnatal day 7 animals and 2 postnatal day 20 animals (n=20) were studied. Isolated outer hair cells from 2 waltzing guinea pigs at postnatal day 7 (n=10), 1 animal at postnatal day 10 (n=19), and 4 animals from postnatal day 20 (n=40) were compared
with control cells. All outer hair cells, from all groups of guinea pigs, responded with an oscillatory length variation following the 10 Hz alternating sinusoidal current. Oscillatory movements were optimal when the electrode position was on the long axis of the cell. The length changes were graded with the stimulus intensity such that increases or decreases in the stimulus intensity were directly followed by a corresponding increase or decrease in the magnitude of movement. Maximal responses of up to 2 µm were obtained when cells were stimulated with the maximal stimulation of a 3 V peak-to-peak signal. No differences in the magnitude or character of the length changes for the different groups were evident. Even cells from the waltzing guinea pig demonstrating swollen cuticular plates, giant stereocilia and slender cell bodies, responded to electrical stimulation in an oscillatory fashion. These cells from the waltzing guinea pig did not show any unusual motile responses to electrical stimulation. Visual detection thresholds were obtained for control and waltzing guinea pig outer hair cells but there were found to be no significant differences between the two groups (Table 2); neither were there differences in the magnitude nor the character of the length changes of the cells. DISCUSSION The results reported here show that at a time when the waltzing guinea pig has a moderate hearing loss: (i) the actin-bearing structures in the organ of Corti (stereocilia, cuticular plate, and subsurface cisternae) show disorganization; (ii) the outer hair cells do not respond with a length change when the intracellular calcium is raised; (iii) electrically-induced motile responses are equivalent to those of control cells; and (iv) passive outer hair cell length changes are induced under hypo-osmotic conditions. The outer hair cells from the waltzing guinea pig allow separations between the slow, metabolically-dependent length changes induced by chemical stimulation and the fast, metabolically-independent length changes induced by electrical stimulation. A similar dissociation has been previously found after in vivo noise exposure (Décory et al., 1991). The slower, chemically-induced type of outer hair cell motility is presumably dependent upon the interaction between calcium and actin/myosin, and the subsequent activation of second messenger systems (Zenner, 1986; Schacht and Zenner, 1986). The inadequate response of the waltzing guinea pig outer hair cells to increases in intracellular calcium may be associated with one of the following sites: (1) intracellular calcium stores, (2) compliance of the plasma membrane, (3) actin/myosin, or (4) second messenger systems. With regard to the lack of a calcium-induced motility, the fluo-3 experiments reveal that ionomycin causes a similar rise in intracellular calcium to that in the control cells. As a consequence, the plasma membrane of the waltzing guinea pig outer hair cell presumably has similar permeability features to those of normal cells. In addition, the osmolarity experiments performed on the outer hair cells also indicate that the compliance of the plasma membrane is not disrupted in the waltzing guinea pigs, since their cells have the potential
Waltzing guinea pig outer hair cells 1143 for passive length changes when the osmolarity of the surrounding medium is reduced. Passive length changes induced by reducing the osmolarity have previously been shown to be independent of extracellular calcium (Dulon et al., 1988). The outer hair cells from the waltzing guinea pig have the capacity for passive length changes. As a result, these hair cells cannot be assumed to be rigid cylinders; rather, they are able to undergo length changes, albeit passive. The waltzing guinea pig exhibits obvious defects in the cell structures known to contain high concentrations of actin. The cuticular plates are swollen, giant stereocilia are evident and, as presented here, the subsurface cisternae are swollen and disorganized. It remains to be determined what role the disorganized actin plays in inhibiting outer hair cell length changes. Although doubtful, it cannot yet be discounted that the bulging cuticular plate, or the giant stereocilia, are contributing to the lack of a motile response in the waltzing guinea pig outer hair cells. Nevertheless, outer hair cells from waltzing guinea pigs that had neither swollen cuticular plates, nor giant stereocilia, were unable to respond with a length change when the intracellular calcium concentration was increased. A host of biochemical reactions that give rise to length changes may also be affected in this mutant guinea pig. Biochemical studies are needed in order to determine which, if any, of the reactions underlying the motile response in outer hair cells would be defective. To date, there are no known reports on the biochemistry of the outer hair cells of the waltzing guinea pig. It is of interest that the isolated outer hair cells from the waltzing guinea pig responded to electrical stimulation. The electrically-induced length changes are neither dependent on actin/myosin interactions, nor on cellular metabolism (Kachar et al., 1986; Ashmore, 1987). It has been shown that, after treatment with trypsin, which digests the cortical lattice in outer hair cells, electromotility is maintained (Kalinec et al., 1992). The sub-cortical lattice has been proposed to maintain the cylindrical shape of the cell and to ensure that the force produced in the membrane is converted into a length change. These findings are not in disagreement with the behavior of the waltzing guinea pig outer hair cells. The disorganization of the subcortical lattice in the mutant cells is followed by a concomitant loss of the calcium-induced motility. Since the electromotility is unaffected in the mutant cells it is suggested that the force generator in the plasma membrane is intact. Finally, it should be mentioned that recent studies (Evans et al., 1991; SantosSacchi, 1992) raise fundamental questions on the physiological significance of outer hair cell electromotility. In contrast to the intact cochlea, the outer hair cells appear to have a poor frequency response and, because of the capacitance of the membrane filter, there is a frequency limit to the mechanical response above several kilo-Hertz. This work was supported by The Swedish Medical Council, The Foundation Tysta Skolan, Torsten and Ragnar Söderberg Foundation, and Hörselframjandet. We wish to thank Dr Jochen
Schacht for his critical reading of the manuscript and Agneta Viberg for expert technical assistance.
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