Structural and ultrastructural aspects of isolated immature cochlear outer hair cells maintained in short-term culture

ELSEVIER

Hearing Research 88 (1995) 169-180

HFdRIrlG RE3FRa.I

Structural and ultrastructural aspects of isolated immature cochlear outer hair cells maintained in short-term culture Marc Lenoir *, Chantal Ripoll, Philippe Vago INSERM U254, and Universit~ Montpellier L CHU H@ital, St. Charles, 34295 Montpellier, France Received 31 May 1994; revised 6 January 1995; accepted 4 May 1995

Abstract

Immature outer hair cells (OHCs), isolated from developing rat cochlea without using proteolytic enzymes, were maintained in short-term culture in a clot of coagulated plasma. Cell viability was assessed by a laser scanning image cytometer, using double-fluorescent labeling. Light and transmission electron microscopy was used to study the morphology of isolated cells. Ten to 60 healthy OHCs were obtained from one cochlea, either as single isolated cells or clusters containing 2-10 cells from the same row. Although dead cells were observed only 1 h after dissociation, there were still viable cells after 6 h. Isolated OHCs were not perfectly cylindrical, due to the immaturity of their cortical structures. One hour after dissociation the ultrastructural organization of the isolated ceils was generally welt preserved, but this was followed by dilatation of the Golgi apparatus and endoplasmic reticulum. Specific changes in isolated OHCs were also observed at the subsurface cisternae and cuticular plate. Although degenerating OHCs generally showed a classic pattern of necrosis, certain morphological features reminiscent of apoptosis were also observed. This study emphasises the difficulty involved in investigating isolated immature OHCs in vitro and provides a basis for future research into the physiological requirements of isolated immature OHCs. Keywords: Isolated outer hair cell; Cochlea; Development; Transmission electron microscopy; Laser scanning image cytometry; Rat

1. I n t r o d u c t i o n

Two different types of cells are involved in the transduction of mechanical stimuli in the cochlea, the inner (IHCs) and outer (OHCs) hair cells. The IHCs are flaskshaped cells with free-standing stereocilia at their apical pole, and are closely surrounded by supporting cells and neighbouring IHCs. Their basal poles are connected by afferent neurons representing 95% of the total afferent supply to the cochlea (Spoendlin, 1969; Pujol and Lenoir, 1986; Eybalin, 1993). IHCs are considered to be true sensory cells in the cochlea (Dallos, 1986). By contrast, OHCs are cylindrical, and their tallest stereocilia are anchored in the overlying tectorial membrane (Lim, 1986). The OHC lateral plasma membranes is in contact with a perilymphatic compartment (spaces of Nuel), and the OHC basal pole is wrapped by Deiters cells (Lim, 1986). OHCs are connected by few afferent neurons, but receive a rich efferent innervation (Spoendlin, 1969; Pujol and Lenoir, 1986; Eybalin, 1993). OHCs are considered to be sensory-

* Corresponding author: Tel.: (33) 6733-6975; Fax: (33) 6752-5601. 0378-5955/95//$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 3 7 8 - 5 9 5 5 ( 9 5 ) 0 0 1 1 0 - 7

effector cells that are responsible for cochlear active mechanisms that control sensitivity and sharp tuning (Davis, 1983; Neely and Kim, 1983). Methods have been recently developed for isolating adult cochlear OHCs in different animal species and for maintaining them in short-term culture (Goldstein and Mizukoshi, 1967; Zajic and Schacht, 1987). These methods have considerably helped to elucidate the functional activity of these cells. Isolated adult OHCs have motile properties that can be separated into two functionally distinct categories: fast and slow motility (Dulon and Schacht, 1992). It has been postulated that fast mechanical responses resulting from OHC electromotile properties (Brownell et al., 1985; Kachar et al., 1986; Dallos et al., 1993; Ashmore, 1987) constitute a mechanical amplification process operating at acoustic frequencies. Slow calcium-dependent motility (Zenner, 1986; Flock et al., 1986; Dulon et al., 1990) is believed to modulate cochlear mechanics by efferent control (Mountain, 1980; Siegel and Kim, 1982; Puel and Rebillard, 1990). There have been many in vitro studies on isolated adult OHCs, but only two studies have investigated isolated OHCs during the period of their maturation. These two

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studies w e r e c o n d u c t e d only recently, and investigated the onset o f both fast and slow motilities o f O H C s in guinea pig e m b r y o s (Pujol et al., 1991) and in n e w born gerbils (He et al., 1994). T h e results o f the studies clearly d e m o n strated that in vitro studies on isolated i m m a t u r e O H C s p r o v i d e a v a l u a b l e insight into certain d e v e l o p m e n t a l aspects o f their p h y s i o l o g y . The present study has two main objectives. The first is

to define appropriate conditions for isolating and maintaining i m m a t u r e rat (Wistar) O H C s in short-term culture. The rat has been used for m a n y years as an e x p e r i m e n t a l m o d e l for studying in v i v o the postnatal c o c h l e a r d e v e l o p m e n t in both normal conditions (Wada, 1923; L e n o i r and Puel, 1987a; L e n o i r et al., 1980,1987; Puel and Uziel, 1987; Roth and Bruns, 1992) and in pathological conditions (Carlier and Pujol, 1980; L e n o i r and Pujol, 1980; U z i e l et

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Fig. I. Phase-contrast micrographs of living dissociated immature OHCs after 1 h survival time. a: solitary OHC; b: 2 adjacent OHCs; c: cluster containing 9 OHCs. All OHCs show birefringent lateral plasma membranes, basal nuclei (n) and upright stereocilia (s). Note the stocky appearance of the OHCs in (a) and (b), the W-shaped stereocilia bundles in (b) and (c), the absence of supporting cells between OHCs in (b) and (c). Bar: 10 /~m. d-f: phase-contrast micrographs of living dissociated immature OHCs after 6 h of survival time. Note the W-shaped stereocilia bundles on all OHCs. Note also the general rounding of the cells. Bar: 10 p,m. g-h: phase-contrast micrographs of 2 degenerative adjacent OHCs after 3 h of survival time. Note the absence of membrane refractility, the dilated nuclei. The stereocilia are not distinguishable. Bar: 10 p,m. h: swollen OHC with a thin "ghost like" membrane. The bundle of stereocila appears to be damaged. Bar: 10 /~m. i: example of cell viability assessment using the LIVE/DEADT M Viability/Cytotoxicity Kit on a cluster containing 9 OHCs (1-9) after 6 h of survival time. The laser scanning fluorescent digitised image reveals that the cytoplasm of OHCs 2, 4, 5, 7, 9 are labeled with C-AM while only the nuclei of OHCs 1, 3, 6, 8 are labeled with EthD-l. OHCs 2, 4, 5, 7, 9 are considered as living cells and OHCs 1, 3, 6, 8 as dead cells. Fluorescence intensity is represented by different shades of grey, corresponding to arbitrary units. Bar: 10 p,rn.

M. Lenoir et al. / Hearing Research 88 (1995) 169-180

al., 1983). The rat organ of Corti is very immature at birth (Wada, 1923), whereas that of the guinea pig is almost fully mature (Pujol and Hilding, 1973). The rat pup therefore offers an interesting model for investigating hair-cell physiology prior to the onset of cochlear functioning that occurs postnataily at about day 9 - 1 1 (Crowley and HeppReymond, 1966; Carlier and Pujol, 1978; Puel and Uziel, 1987). The second objective of the study is to characterise the morphology and ultrastructure of isolated cells, using light and electron microscopy.

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remaining non-dissociated organ of Corti was removed from the medium with thin forceps, and 10 /xl of chicken plasma (GIBCO) was carefully added to the drop of Ca2+/Mg2+-free HBSS containing thrombin. Following coagulation of the plasma, the well was filled with 500 /xl of HBSS and kept in the humid chamber at 20°C. After 3 h, the medium was replaced with fresh HBSS to avoid excessive basification ( > 7.4) and hyper-osmolarity ( > 330 mOsm). 2.2. Light microscopy

2. Material and methods Fifty Wistar rats aged 5 - 7 days were used in this study. At this stage o f development OHCs have not attained their definite shape (Roth and Bruns, 1992), are not free-standing in fluid spaces, and their basal poles are only in contact with afferent dendrites (Lenoir et al., 1980). Different experimental procedures were tested to isolate and maintain immature OHCs in short-term culture. These procedures included using proteolytic (pronase or trypsin or dispase) and non-proteolytic enzyme (collagenase), using adhesive substrates (collagen, laminin or poly-L-lysine) and matrixes (Matrigel, coagulated plasma), and incubation in CaZ+/Mg2+-free medium (modified phosphatebuffered saline (PBS) or modified Hanks' balanced salt solution (HBSS). We describe here the simplest method which reproducibly obtained the greatest number of surviving dissociated OHCs with the longest survival time. Data was obtained up to 6 h after cell dissociation, since living isolated immature OHCs rarely survived for longer. 2.1. Cell isolation

Temporal bones were excised from animals under deep anaesthesia (Nembutal, 50 m g / k g ) and immersed in HBSS (GIBCO) at 4°C in a Falcon 35-mm plastic culture Petri dish. The osmolarity o f the medium was previously measured with a vapor-pressure osmometer (Wescor) and adjusted to 300 _ 2 mOsm by addition of glucose. The pH was maintained at 7.2 with 5 m M HEPES. Each cochlea was exposed by removing the cartilaginous otic capsule. The stria vascularis was dissected out and the organ of Corti separated from the modiolus. Ten minutes later, the organ of Corti was transferred into a 10-/xl drop of HBSS without calcium and magnesium (GIBCO), supplemented with thrombin (0.5 m g / m l , Sigma). The drop was previously deposited in the center of a well ( = 10 m m diameter) formed with silicon on a 35-mm round glass coverslip treated with Vectabond (Vector SP 1800) and kept at room temperature (20°C) in a Falcon 60-mm plastic culture Petri dish saturated with water vapor. After 20 min, the drop containing the organ o f Corti was gently passed 5 times through the tip o f a Gilson Pipetman (10 /xl) in order to disperse the OHCs. Cells were left to settle for 5 min. The

The morphological status and the optical characteristics of the isolated hair cells were monitored by iterative examinations of about 10 min each, under a Zeiss inverted light microscope equipped with phase contrast lens, 100 × oil immersion objective, and caloric-absorption filter (Zeiss). Each preparation was observed twice: immediately after plasma coagulation and at a given survival time between 1 and 6 h. In order to protect the sample against contamination and dehydration, the cap of a Falcon 35-mm culture Petri dish was placed over the coverslip. The criteria used to identify OHCs in the plasma clot were shape, nucleus position and stereocilia organisation. 2.3. Assessment o f cell viability

Cell viability was assessed for up to 6 h using the L I V E / D E A D TM Viability/Cytotoxicity Kit from Molecular Probes (L-3224). The kit includes two probes: calcein

Table 1 Live/dead assays Trail

OHCs

Trial i 1h Trial 2 lh Trial 4 3h Trial 4 3h Trial 5 6h Total number

SingleOHCs OHCswithin clusters SingleOHCs OHCswithin clusters SingleOHCs OHCswithin clusters SingleOHCs OHCswithin clusters SingleOHCs OHCswithin clusters SingleOHCs OHCswithin clusters

Green+ Green-/ red 10 2 47 0 5 1 14 0 1 29 0 5 3 32 0 6 5a 0 27 6 127 0

Red + Total number 6 18 0 47 3 9 0 14 5 6 0 29 2 l0 2 32 5 Il 6a Il a 21 54 6 133

Five representative individual assays on isolated OHCs with the LIVE/DEADT M Viability/Cytotoxicity Kit using C-AM (green fluorescence) and EthD-1 (red fluorescence). Cells were counted after 1 h (2 assays), 3 h (2 assays), and 6 h (1 assay) following tissue dissociation. The number of recognisable single OHCs and OHCs within isolated clusters is reported versus the coloration of their fluorescence: green+ (functional cells); red + (dead cells); green/red- (non-functional and EthD-I impairment cells). The number of clusters is indicated in parenthesis. -, not counted. a Green and red OHCs mixed within the clusters.

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M. Lenoiret al. / HearingResearch88 (1995) 169-180 A M (C-AM), a fluorogenic substrate that is cleaved by intracellular esterases only in viable cells to form a green fluorescent m e m b r a n e - i m p e r m e a n t product, and ethidium homodimer-1 (EthD-1), a high-affinity red fluorescent D N A intercalator that is only able to pass through the compromised membranes o f dead cells. L i v e / d e a d assays were performed according to the protocol provided by Molecular Probes in a single step by replacing the 500-/21 HBSS bathing the plasma clot with 500 /xl of HBSS containing approximately 2 /xM C - A M and 4 / x M EthD-1. Cells were incubated for 30 min in the working solution. Then, both green (520 nm) and red (620 nm) fluorescences o f dissociated O H C s were simultaneously quantified by a Laser Scanning I m a g e Cytometer ( A C A S 570 Meridian) operating at 488 nm (excitation wavelength for both probes).

2.4. Preparation for transmission electron microscopy The ultrastructural integrity o f isolated OHCs was investigated using TEM, 1, 3 and 6 h after isolation. Fixation was performed by replacing the HBSS with glutaraldehyde (3.5%) in cacodylate buffer (0.1 M). After 1 h the culture was rinsed with the same buffer (0.2 M, pH 7.2-7.4), the sample postfixed with osmic acid (1% in water) for 1 h, and then washed once again with the buffer. Dehydration was performed in graded series o f ethanol (50% to 100%). For final dehydration, the preparation was processed by critical-point drying with CO 2. Embedding was performed in epon for 60 h at 70°C. Isolated OHCs were localised in the block o f resin under a light microscope, and pieces o f epon containing cells were cut off with a 0.5-mm surgical punch. Semi-thin sections were cut until OHCs were reached. Thin sections, mounted on either 200-mesh or formwar-coated grids and double-stained with uranyl acetate and lead citrate, were observed under an Hitachi 7100 transmission electron microscope.

3. R e s u l t s

3.1. Light microscopy Preparations contained elongated cells and round cells. The elongated cells were identified as hair cells by the

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presence o f a cuticular plate and stereocilia. A number of round cells were classified as damaged hair cells, since they also showed a cuticular plate and stereocilia. A m o n g the other isolated round cells it is probable that there were damaged supporting cells, since neither pillar, Hensen, or Deiters cells were recognizable in the plasma clot. During the first hour after dissociation, it was generally possible to discriminate between immature IHCs and OHCs by using certain morphological criteria that enable both types o f adult isolated hair cells to be distinguished (Dulon et al., 1991). IHCs were pear shaped, whereas OHCs tended to be cylindrical. The neck region of IHCs was smaller than that of OHCs, and severely tilted to one side. The nucleus was located higher in IHCs than in OHCs. Cell length, a reliable criterion for distinguishing between isolated adult IHCs and OHCs, was not used in this study as at this stage o f cochlear development in rats both IHCs and OHCs ranged approximately between 20 and 30 /zm. After a longer survival time it was more difficult to distinguish between IHCs and OHCs, as many OHCs tended to resemble IHCs. In most cases, however, OHCs could be identified by their W - s h a p e d bundles o f stereocilia. He and co-workers (1994) have reported the chance o f mistaking IHCs and OHCs as quite small, since OHCs formed a large proportion o f the isolated hair-cell population. In the present study, there were no more than 2 or 3 isolated IHCs per cochlea. These IHCs were always observed as solitary cells. The immature OHCs were either isolated (Fig. 1a,d,h), or remained in clusters o f up to 10 cells (Fig. l b , c , e - g ) . The amount o f OHCs ranged from 5 to 25 isolated single cells, and the number o f clusters ranged from 1 to 20 per cochlea. Most o f the clusters were made o f OHCs from a same row since cells were in close contact with each other, a normal feature o f OHC early maturation (Roth and Bruns, 1992). It is believed that the isolated cells originated from different regions between the base and apex of the cochlear spiral, since the longest OHCs ( = 30 /zm) generally had the longest stereocilia ( = 4 /zm), and the narrowest W-shaped bundles of stereocilia. Morphological studies on adult isolated OHCs in different animal species (Lim and Flock, 1985; Zajic and Schacht, 1987) report healthy isolated OHCs as having a cylindrical shape, refractile appearance, and a basal nucleus. There is no distortion of the cell plasma membrane,

Fig. 2. Transmission electron micrographs of a cluster of OHCs after 1 h of survival time. a: 3 adjacent immature OHCs. The surrounding grey material corresponds to coagulated plasma. The close apposition of the lateral plasma membranes indicates that OHCs belong to the same row. For all 3 cells, the lateral plasma membranes are regular and stereocilia are upright. Mitochondria are scattered throughout the cell body and the nuclei are in basal position. Both external cells (O1) and (03) have a clear cytoplasm. Their nuclei show slight chromatin condensation. In 03, the cytoplasmic organelles are well preserved. In O1, the endoplasmic reticulum is vesiculated (arrows). The central OHC (02) is slightly out of the cutting plane compared with Ol and 03. 02 shows dark cytoplasm, vesiculated and dilated endoplasmic reticulum (arrows) and dilated subsurface cisternae (arrowheads). Note the large clear areas above the nucleus. The nucleus shows clumped chromatin. The dark material beneath the basal pole of C2 could be cytoplasmic extrusion from the cell. Bar: 2.5 /xm. b: high magnification at the level of the lateral plasma membranes of 02 and 03 in the supra nuclear region. In 03, organelles appear well preserved. One row of well-defined subsurface cisternae (arrowheads) lines the lateral plasma membrane. Note the absence of pillars between lateral plasma membrane and subsurface cisternae. In 02, subsurface cisternae (arrowheads) and endoplasmic reticulum (arrows) are dilated. Note the abnormally high density of free ribosomes in the cytoplasm and the clear area (asterisk) devoid of other organelles. Bar: 0.5 p,m.

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Fig. 4. Transmission electron mlcrographs of 2 isolated OHCs after 6 h of survival time. a: round OHC at one extremity of a cluster, showing a large cytoplasmic protrusion (star), a vacuolised cytoplasm and dense chromatin masses. Note the normal appearance of the stereocilia and the presence of links between them (arrows). Bar: 1.5 /xm. b: immature isolated OHC showing advanced necrosis. A piece of cuticular plate (cp) is recognisable at the apex of the cell. Both the lateral cell membrane and the nuclear envelope (he) are greatly altered. Bar: 2.5 /xm.

no p r o t r u s i o n o f c y t o s o l , n o r B r o w n i a n m o t i o n o f the c y t o p l a s m i c o r g a n e l l e s . In o u r study, O H C s m e e t i n g t h e s e criteria (although not perfectly cylindrical) were observed up to 6 h a f t e r d i s s o c i a t i o n . P r e p a r a t i o n s also c o n t a i n e d O H C s t h a t h a d lost t h e i r b i r e f r i n g e n c e a n d t h a t w e r e g r a n u l a r in a p p e a r a n c e . T h e s e d e g e n e r a t i n g cells w e r e m o r e o r less r o u n d a n d o f t e n e x h i b i t e d a d i l a t e d n u c l e u s (Fig. lg). In a d d i t i o n , s w o l l e n O H C s w i t h a t h i n ' g h o s t - l i k e ' m e m b r a n e (Fig. l h ) w e r e o b s e r v e d , in a g r e e m e n t w i t h previous descriptions of adult isolated OHCs (Lim and F l o c k , 1985). In t h e s e d e g e n e r a t i n g cells, m i t o c h o n d r i a

w e r e o b s e r v e d to h a v e r a p i d B r o w n i a n m o t i o n s . B o t h h e a l t h y a n d d e g e n e r a t i n g O H C s w e r e p r e s e n t after 6 h, although there were more degenerating OHCs, and a larger n u m b e r o f cells w e r e r o u n d . 3.2. Viability a s s e s s m e n t

L i f e / d e a d a s s a y s f r o m 7 trials p e r f o r m e d at d i f f e r e n t t i m e s d u r i n g a 1 - 6 h p e r i o d are s h o w n in T a b l e 1. W h a t e v e r t h e d e l a y a f t e r isolation, O H C s c o u l d b e d i v i d e d in 3 g r o u p s a c c o r d i n g to t h e i r s t a i n i n g p a t t e r n : (1) O H C s

Fig. 3. Transmission electron micrographs of isolated OHCs after 3 h of survival time. a: apical pole of a single isolated OHC. Two cytoplasmic protrusions (stars) are visible at the level of the cutieular plate (cp). Note dilation of Golgi apparatus (G) and vacuolisation of mitochondria (large arrows). Links between stereocilia are distinguishable (small arrows). Bar: 1.5 /xm. b: detail of a cytoplasmic protrusion emerging near the kinocilium (k) of an isolated immature OHC. Note the presence of many uncoated vesicles (arrowheads) of various sizes within the protrusion. Bar: 0.25 /xm. c: high magnification of the dilated Golgi apparatus visible on micrograph (a). Bar: 0.5 /~m. d: basal pole of an immature isolated OHC. Unvesiculated afferent endings of various size (a) with resealed lateral membrane are attached to the OHC. The largest one is almost devoid of cytoplasmic content. The surrounding cellular material probably corresponds to remnants of Deiters cells (rD). Bar: 0.5 /~m. e: detailed view of a contact between the base of an immature isolated OHC and a resealed afferent ending (a). Note the presence of 2 coated pits (arrowheads) at the presynaptic OHC membrane facing the afferent. Bar: 0.25 /~m.

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with a green cytoplasm (C-AM stained), (2) OHCs with a red nucleus (EthD-1 stained), and (3) OHCs with neither a green cytoplasm nor a red nucleus. OHCs in the first group were considered as functional cells due to their intracellular esterase activity. OHCs in the second group were considered as dead cells due to the permeability of their membranes. OHCs in the third group were considered as non-functional cells with impermeant plasma membranes. Although dead OHCs were observed after only 1 h, some OHCs were still live 6 h after dissociation (Fig. l i). Within the clusters, there were generally more surviving cells present than dead cells. There was no obvious difference in the case of single OHCs.

pole of the isolated OHCs, the lateral membrane often showed numerous coated pits (Fig. 3d), whilst the cytoplasm displayed a large number of coated vesicles. A few hours after dissociation, a number of isolated OHCs were observed to be round in shape (Fig. 4a). Stereocilia were mostly normal in appearance, with no obvious signs of fusion (Fig. 4a). Side-to-side and row-torow links (Pickles et al., 1984) were frequently observed (Fig. 3 and Fig. 4a), and tip links (Pickles et al., 1984) were occasionally present (picture not shown). After longer periods in the plasma clot, degenerating OHCs showed extensive cytoplasmic vacuolization and disrupted lateral plasma membrane, with extrusion of intracellular content (Fig. 4b).

3.3. Transmission electron microscopy

Immature OHCs with a well-preserved ultrastructural organization were observed 1 h after dissociation (Fig. 2a). These OHCs had a smooth lateral membrane, bordered by a single layer of well-defined subsurface cisternae (Fig. 2b). At this stage of development there were no pillars or arms between the lateral membrane and subsurface cisternae, as is the case in adult OHCs (personal observations). Mitochondria were dispersed throughout the cytoplasm (Fig. 2a), as seen during the early stage of in vivo OHC development (Weaver and Schweitzer, 1994). The cristae of the mitochondria, Golgi apparatus, and endoplasmic reticulum were well preserved (Fig. 2b). The membrane, the core and the rootlets of the stereocilia had a normal aspect (Figs. 2 and Fig. 3a). Dispersed chromatin was observed in the nucleus. Dilation of the Golgi apparatus and vesiculation of the endoplasmic reticulum (Figs. 2a,b and 3a,c) was a commonly found cytoplasmic abnormality. Vacuolization without swelling of mitochondria was also frequently observed in these stressed cells (Fig. 2a,b). There was also swelling of the subsurface cisternae in some of the damaged cells, as well as the formation, in the middle of the cell, of large and clear areas not limited by a membrane, but filled with what appeared to be free ribosomes (Fig. 2a,b). Degenerating cells often had an increased electron density, due to accumulation of free ribosomes within the cytosoi (Fig. 2a,b). Cytoplasmic protrusions without membrane rupture and filled with numerous coated and uncoated vesicles were frequently observed at the cuticular plates (Figs. 3a,b and 4a). Convolution of the nuclear outline (figure not shown) and compaction of chromatin into dense masses were also observed (Fig. 2 and Fig. 4a). Broken unvesiculated nerve endings with resealed lateral membrane, 0.1- and 1-p.m diameter, were frequently found attached to the basal pole of isolated OHCs (Fig. 3d,e). The largest buttons often displayed very little cytoplasmic material. Larger masses of cellular remnants, 5 - 8 /~m diameter, were also occasionally observed at the basal pole of the isolated OHCs. These masses probably corresponded to disrupted Deiters cells (Fig. 3d). At the basal

4. Discussion The data in this study show that viable immature OHCs can be isolated from the organ of Corti of 5-7-day-old rat pups and be maintained in short-term culture in a plasma clot for severafhours. The study also provides information concerning the ultrastructural modifications accompanying the progressive degeneration in culture. 4.1. Experimental procedures

Although enzymes efficiently dissociate adult (Goldstein and Mizukoshi, 1967; Lim and Flock, 1985) and immature OHCs (Pujol et al., 1991; He et al., 1994; personal unpublished results), they were not used in this study. Enzymatic dissociation requires several rinses, dramatically reducing the initial number of isolated hair cells. What is more, treatments with proteolytic enzymes such as pronase (Goldstein and Mizukoshi, 1967) and trypsin (Ulfendahl and Slepecky, 1988) can damage the cells (Zenner et al., 1985). Both calcium-independent and calcium-dependent cell adhesion proteins, NCAM (Whilt and Rutishauser, 1990) and E-cadherin (Whitlon, 1993), respectively, are present at the surface of OHCs during cochlear development in the mouse. We therefore pre-incubated the tissues in a C a 2+and MgZ+-depleted medium to facilitate cell isolation. This procedure clearly produced more isolated viable OHCs than the same procedure with CaZ+/Mg2+-containing medium (unpublished observations). Conversely, the absence of E-cadherin from immature IHCs (Whitlon, 1993) could help to explain why very few of them were isolated in our study. On the other hand, no intact isolated supporting cells were recognizable in the plasma clots, even though E-cadherins are present at the surface of the immature pillar and Deiters cells (Whitlon, 1993). This suggests that these immature cells are highly sensitive to mechanical constraints a n d / o r have strong attachments with the basilar membrane. Preparation of isolated OHCs for histology and for

M. Lenoiret al. /Hearing Research 88 (1995) 169-180

immunology is difficult under standard conditions, owing to the weak adhesion of cells to the supporting surface. To date, collagen gel has been used to hold isolated OHCs during addition of fresh medium, and during histological preparations (Slepecky and Ulfendahl, 1988). Recently, collagen gel was also used to maintain organotypic preparation of rat cochlea in culture (Rastel et al., 1993). In the present study, we have used a very simple plasma embedding procedure (G~ihwiller, 1981). This procedure offers the same advantages as a collagen gel with regards to light microscopic observations and histological preparations for TEM. Furthermore, the plasma clot is well adapted to functional investigations using various fluorescent probes (personal observations), and to treatment of cultured cells with trophic or growth factors (Gueritaud and Seyfritz, 1992). Nevertheless, some exogenous trophic a n d / o r growth factors could be provided by the plasma clot itself. 4.2. Yield of immature isolated OHCs

After isolation, the yield of immature OHCs varied from 10 to 60 cells per cochlea. Compared with adult guinea pig or chinchilla, this number appears to be rather low, since a single cochlea from these animals can provide several hundred OHCs (Zajic and Schacht, 1987). However, the fact that immature OHC are in close contact with both each other and Deiters cells could make them more difficult to separate than mature cells, that are free-standing in the spaces of Nuel (Pujol et al., 1991). Anatomical differences between species could also contribute to the limited yield of isolated immature OHCs in rat cochlea. Adult rats, mice, and gerbils give a considerably lower number of cells than adult guinea pig and chinchilla (Zajic and Schacht, 1987). It is noteworthy that cell connections between the OHCs and their supporting cells, the pillars of Corti and the Deiters cells, are far more developed in cochlea that specializes in high-frequency reception (Vater and Lenoir, 1992; Vater et al., 1992) than in cochlea that specializes in low-frequency reception (Raphael et al., 1991). Rats, mice and gerbils, which provide a limited yield of isolated OHCs, have higher hearing frequency ranges than guinea pig and chinchilla (Pujol et al., 1992a). 4.3. Morphological features of well-preserved isolated immature OHCs

This study compares the morphology of well-preserved isolated OHCs with that of OHCs conventionally prepared from whole rat organ of Corti at the same stage of development (Lenoir et al., 1980; Roth and Bruns, 1992). The main body of the immature OHCs was observed to have a large cytoplasmic concentration of mitochondria, endoplasmic reticulum, Golgi bodies and ribosomes. Similar observations have been made on young gerbil cochlea (Weaver and Schweitzer, 1994). In 5-7-day-old Wistar rats, young OHCs have not attained their full length. They

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are not perfectly cylindrical, having a rather stocky appearance (Lenoir et al., 1980; Roth and Bruns, 1992). In adult rats the cylindrical shape of the OHCs is maintained by the cortical lattice, a 2-dimensional actin-based structure that lies beneath the lateral plasma membrane (Hoiley and Ashmore, 1988,1990; Slepecky, 1989; Holley et al., 1992) and by the subsurface cisternae, one or several rows (1 row in rats) of concentric endoplasmic membranes lining the cortical lattice (Saito, 1983; Flock et al., 1986; Forge, 1991; Bannister et al., 1988). In 5-7-day-old Wistar rats the subsurface cisternae are present, but the pillars which link the cortical lattice to both the subsurface cisternae and the lateral plasma membrane in adult OHCs (Forge, 1986, 1991; Raphael and Wroblewski, 1986) are not formed (present study). The absence of the pillars could therefore influence the shape of the isolated immature OHCs. Interestingly, a recent gerbil study reported that actin labelling in the region of the cortical cytoskeleton significantly increased during the onset of the gross cochlear potentials (Weaver et al., 1994). This finding suggests that the cortical lattice is not fully developed in 5-7-day-old Wistar rats, as the onset of the cochlear function occurs postnatally after 9-10 days in this animal (Crowley and Hepp-Reymond, 1966; Carlier and Pujol, 1978; Puel and Uziel, 1987). In the present study, unvesiculated nerve endings with resealed lateral membrane were frequently observed at the basal pole of isolated OHCs. The nerve endings were probably afferent, since at this stage of development the basal pole of the OHCs only forms synapses with afferents (Lenoir et al., 1980). The largest ones contained very little cytoplasmic material, similar to the swollen afferent dendrites observed under IHCs after either acoustic overstimulation (Robertson, 1983) or ischemia-related excitotoxicity in adult animals in vivo (Pujol et al., 1992b; Puel et al., 1994). Furthermore, numerous coated pits and coated vesicles were observed at the basal pole of the isolated cells. In neurons and sensory cells, endocytosis contributes to the recycling of presynaptic membrane created by synaptic vesicle fusion (Pley and Parham, 1993). Isolated immature OHCs with afferent endings could thus provide a useful model for studying transient afferent synapses of developing OHCs. It has been proposed that the transient afferent dendrites under OHCs form part of the radial system, since they react in the same way to kainic acid exposure (Pujol et al., 1985). 4.4. Morphological features of the degenerating isolated OHCs

Although there were some individual variations in morphological features accompanying degeneration of isolated immature OHCs, there was evidence for a general pattern of necrosis characterized by vesiculation of the cytosol and alteration of the cell wall. Pathological features specific to particular OHC anatomy were also observed, namely

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changes at the subsurface cisternae and formation of apical cytoplasmic protrusions. The initial swelling of Golgi apparatus and endoplasmic reticulum was often accompanied by dilatation at the subsurface cisternae. Indeed subsurface cisternae, that have certain characteristics in common with both the Golgi apparatus and the endoplasmic reticulum (Pollice and Brownell, 1993; Weaver and Schweitzer, 1994), are highly vulnerable to a variety of influences (Evans, 1990; Forge et al., 1993; Dieler et al., 1991). Cytoplasmic protrusions of the cuticular plate of hair cells are commonly attributed to hypoxia or xenobiotic intoxication in the whole organ of Corti in vivo (Lavigne-Rebillard and Pujol, 1986; Lenoir and Puel, 1987b), and in cochlear organotypic cultures (Forge and Richardson, 1993; Richardson and Russell, 1991; Kotecha and Richardson, 1994). The presence of many uncoated vesicles at the apical pole of the isolated OHCs suggests that their fusion with the plasma membrane led to the formation of the protrusions. Such a massive exocytosis in isolated immature OHCs could be the consequence of small increases in intracellular pressure (Finkelstein et al., 1986; Zorec and Tester, 1993) due to failing ionic pumps. It is also possible that such a phenomenon could influence the shape of isolated immature OHCs, by provoking cytoplasmic leakage within apical protrusions. Some isolated immature OHCs had morphological features reminiscent of apoptosis (Wyllie, 1981): dense chromatin masses appearing within the nucleus, and increase in electron density of the cytoplasm. It is thus possible that apoptosis occurs in OHCs in certain environmental conditions. 4.5. Possible causes of cell degeneration Mechanical trauma occurring during cell preparation is a decisive factor in adult isolated OHC survival (Goldstein and Mizukochi, 1967; Zajic and Schacht, 1987). Dissection of the organ of Corti a n d / o r flux and efflux of the tissue through the pipette probably caused mechanical trauma to a number of immature OHCs, since dead cells with ruptured lateral plasma membrane were observed soon after dissociation. In vivo, OHCs are attached to Deiters cells by lateral attachments at the apical junctional complexes (Beagley, 1965; Forge, 1986). Immature OHCs are attached all along their lateral sides to each other and to Deiters cells (Lenoir et al., 1980; Roth and Bruns, 1992). It is therefore possible that the risk of lateral plasma membrane injury during isolation is potentially greater for immature than for adult OHCs, since the lateral plasma membrane of the latter is free from any contacts. On the other hand, stereocilia tufts on isolated immature OHCs appeared to neither suffer mechanical damage as a result of the dissociating procedure, nor biochemical stress despite the fact that the balance in Na ÷ and K + of the culture medium was different from that of the endolym-

phatic fluid already containing high K ÷ and low Na + concentrations at this stage of development (Bosher, 1972). In this study a number of isolated OHCs were observed to have abnormal mitochondria, indicative of hypoxia. In adult guinea pigs and rats, the cochlear structures use glycolysis for maintaining normal activity during transient hypoxia (Lotz et al., 1977). It is not known whether immature hair cells can also use such an anaerobic pathway. It is interesting to note that the developing organ of Corti is richly vascularized by a large spiral vessel, which by the end of the cochlear maturation regresses and even degenerates in animals such as rats and gerbils, (Lenoir et al., 1980; Axelsson et al., 1986). Immature OHCs could therefore be more dependent on a direct oxygen supply than adult ones. In vitro, cell degeneration can also result from decreased metabolism when the culture medium lacks essential nutriments. The standard culture medium used in this study, a HBSS, was similar to that commonly used for preparing isolated adult OHCs (Bobbin et al., 1990), and was devoid of any other substances required for normal and prolonged cell metabolism. It might be necessary to supplement the culture medium with adequate components in order to reduce causes of physiological stress, and promote cell survival. Substances which might be required in the early development of the cochlear hair cells could be added, such as thyroxin (Uziel et al., 1983), retinoic acid (Kelley et al., 1993), and neurotrophic factors. It is possible that immature isolated OHCs in vitro are deprived of certain vital interactions with cells that could produce neurotrophic factors. Afferent neurones, for instance, are believed to neurotrophically influence the normal differentiation and maintenance of cochlear hair cells (Gil-Loyzaga and Pujol, 1987; Pujol and Lavigne-Rebillard, 1992).

Acknowledgements We are indebted to Dr. M. Recasens and to the members of his group for introducing the culture technique to us, providing helpful assistance and suggestions in the first stages of this study. We are deeply grateful to Dr. J.P. Gueritaud and to N. Seyfritz for valuable discussion and advice on the use of the plasma embedding procedure. Many thanks are due to Drs K.C. Horner, J. Schacht and J. Fessenden and to researchers of our laboratory, including Pr. R. Pujol for critical comments on the manuscript. We wish to thank P. Sibleyras for invaluable photographic work and M. Margout and P. Paulet for expert technical assistance.

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