Distribution of immunophilin FKBP-12 protein and mRNA within the mammalian cochlea and cochlear nucleus.

Immunophilin FK binding protein-12 (FKBP-12), the soluble receptor for the immunosuppressant drug FK506, is involved in a number of neuronal activities including increased nerve regeneration in the peripheral nervous system and enhanced recovery in animal models of neurodegenerative diseases. In addition, FKBP-12 is tightly bound to the calcium release channel ryanodine receptor and physiologically interacts with the inositol 1,4,5-trisphosphate receptor. In nearly all cell types, release of intracellular Ca(2+) and subsequent second messenger signaling involves activation of these ion channels. We determined the distribution of FKBP-12 within the mammalian cochlea and dorsal cochlear nucleus (DCN) in order to gain insight into Ca(2+) regulation within the cochlea and to possibly identify potential cellular targets for neuroimmunophilin ligands that may prove useful in protection and recovery following ototoxic insult. FKBP-12 protein and mRNA were found to be abundant throughout rat and guinea pig cochlea and DCN.

Overexpression of copper/zinc-superoxide dismutase protects from kanamycin-induced hearing loss.

The participation of reactive oxygen species in aminoglycoside-induced ototoxicity has been deduced from observations that aminoglycoside-iron complexes catalyze the formation of superoxide radicals in vitro and that antioxidants attenuate ototoxicity in vivo. We therefore hypothesized that overexpression of Cu/Zn-superoxide dismutase (h-SOD1) should protect transgenic mice from ototoxicity. Immunocytochemistry confirmed expression of h-SOD1 in inner ear tissues of transgenic C57BL/6-TgN[SOD1]3Cje mice. Transgenic and nontransgenic littermates received kanamycin (400 mg/kg body weight/day) for 10 days beginning on day 10 after birth. Auditory thresholds were tested by evoked auditory brain stem responses at 1 month after birth. In nontransgenic animals, the threshold in the kanamycin-treated group was 45-50 dB higher than in saline-injected controls. In the transgenic group, kanamycin increased the threshold by only 15 dB over the respective controls. The effects were similar at 12 and 24 kHz. The protection by overexpression of superoxide dismutase supports the hypothesis that oxidant stress plays a significant role in aminoglycoside-induced ototoxicity. The results also suggest transgenic animals as suitable models to investigate the underlying mechanisms and possible strategies for prevention.

Glial cell line-derived neurotrophic factor. Potential for otoprotection.

Sensorineural hearing loss results from the degeneration of hair cells and/or auditory neurons in the cochlea of the inner ear. BDNF and NT-3 were shown to support survival of auditory neurons both in vitro and in vivo. Cochlea from P3-P4 rats were cultured as floating explants and hair cells in the organ of Corti were identified by phalloidin-FITC immunostaining. Treatment with cisplatin (35 micrograms/mL) or neomycin (0.6 mM) resulted in 21.2 +/- 6.0% and 7.4 +/- 4.7% surviving hair cells, respectively, after 3 days in culture. GDNF, added together with the ototoxins, increased their number to 46.7% and 37.4%, respectively. In cultures of dissociated cochlea from 4-week-old rat, cisplatin (5 mg/mL) added 24 h after seeding resulted in only 6.1 +/- 1.2% surviving neurons. However, when cisplatin was added together with GDNF (10 ng/mL), 32.8 +/- 1.0% of the neurons survived. The efficacy of GDNF in animal models of ototoxicity was tested next. Guinea pigs were pretreated with GDNF in one ear, delivered either by infusion into the inner ear (scala tympani) with Alzet minipumps (50 ng/mL at a 0.5 microL/h), or injected into the middle ear (120 microL at 1 mg/mL) through the tympanic membrane. The ear that did not receive GDNF always served as control. Ototoxicity was induced systemically either by intraperitoneal cisplatin injections (1 mg/kg/day for 15 days or two injections of 7.5 mg/kg at a 5-day interval or by a combination of kanamycin (200-300 mg/kg, administered subcutaneously) and ethacrinic acid (40 mg/kg, intravenous). It was found that the number of surviving hair cells in GDNF-treated ears was about twice that of control ears in animals exposed to the ototoxins. The transducing GDNF receptor (ret) is expressed in the inner ear.

Living isolated cells from inner ear vessels: a new approach for studying the regulation of cochlear microcirculation and vascular permeability.

The spiral modiolar artery with its proximal branches and the microvessels in the spiral ligament and the stria vascularis were microdissected from the guinea pig cochlea. After incubation with proteolytic and collagenolytic enzymes the mixed cell suspension was fractionated by gradient centrifugation. The cells migrated according to their buoyant densities into the fractions of 1.04 g/ml (endothelial cells), 1.06 g/ml (vascular smooth muscle cells obtained from the spiral modiolar artery; strial pericytes) and 1.08 g/ml (pericytes obtained from the spiral ligament). To test for viability cells were loaded with a fluorescent vital stain (BCECF-AM); for identification, cell-specific stains were used. Identity of endothelial cells (ECs) was confirmed using acetylated low density lipoprotein fluorescently labeled with dioctadecyl-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Pericytes were identified immunofluorescently using the method according to Nayak et al. (1988). Vascular smooth muscle cells were stained for F-actin with rhodamin-phalloidin. This in vitro technique may open new approaches to study local factors involved in microcirculation and vessel permeability of various cochlear vascular beds.

Membrane stains as an objective means to distinguish isolated inner and outer hair cells.

The use of isolated cochlear outer and inner hair cells has become widespread. While the morphological features of these two cell types in general are sufficiently different to allow discrimination, there are situations where confusion can arise. Small outer hair cells, particularly when they are swollen or distorted, can take on an appearance suggestive of inner hair cells. We describe here two fluorescent membrane stains, 3,3'-dihexyloxacarbocyanine iodide and rhodamine B hexyl ester, as an objective means to distinguish between cochlear hair cell types. Both stains mark the subsurface cisternae of outer hair cells thereby delineating the cell outline, and the interior of the cell shows discrete structure. On the other hand, in inner hair cells, the outline of the cell is not resolved while the interior is diffusely fluorescent. Since the two probes have different excitation and emission wavelengths (fluorescein- and rhodamine-like, respectively), this staining procedure can even be used in the presence of another fluorescent marker (for example, a calcium-indicating dye) by appropriate choice of the membrane stain.

Structural variability of the sub-surface cisternae in intact, isolated outer hair cells shown by fluorescent labelling of intracellular membranes and freeze-fracture.

The intracellular membrane systems in intact, isolated outer hair cells were visualised using the fluorescent membrane probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and by freeze-fracture, and f-actin distribution was examined with rhodamine-phalloidin. DiOC6 stained the sub-surface cisternal membranes in the lateral wall and revealed a membrane system running in the centre of the cell from the nucleus to the sub-cuticular region. In optical sections of the lateral wall of fluorescently labelled cells, obtained by scanning laser confocal microscopy, the sub-surface membrane appeared as a fenestrated sheet or a fine network of tubules. Freeze-fracture replicas of rapidly-frozen, unfixed outer hair cells also showed the sub-surface membrane as a fenestrated sheet in some cells or as a network of tubules in others. These combined studies indicate that the interruptions within the cisternal membranes as seen in normal thin sections of outer hair cells are not fixation artefacts but may reflect the dynamic and plastic properties of this membrane system. Double staining of cells with rhodamine-phalloidin and DiOC6 showed substantial co-localisation of intracellular membranes and f-actin. The results suggest there may be a continuous, dynamic endoplasmic reticulum system, forming a core in the centre of the cell, broadening in the subcuticular region and extending down the lateral wall, that may have a role in the turnover and distribution of cytoskeletal assemblies within the outer hair cell.

Scanning electron microscopy of the mammalian organ of Corti: assessment of preparative procedures.

Different fixation, drying and coating procedures have been applied in preparation of the organ of Corti for scanning electron microscopy (SEM), and structural features of the apical surface of the tissue in unfixed, freeze-fractured preparations used in assessing their effects on morphology. Fixation with glutaraldehyde alone or osmium tetroxide alone causes artefacts that are substantially avoided when tissue is doubly fixed in glutaraldehyde followed by osmium. Significant improvements in preservation are also obtained when tissue is additionally processed through thiocarbohydrazide-osmium (TOTO) processing. In addition to providing a conducting coat, it stabilises the tissue against deformations that might otherwise occur during drying, and reduces the extent of tissue shrinkage. Freeze-drying of TOTO processed tissue produces less tissue distortion than critical point drying (CPD) but is not so easy to apply routinely. The distortions of structure in TOTO-processed CPD tissue are not significant and this may be the preferred procedure for routine use, but air drying from hexamethyldisilazane is a useful alternative, producing results as good as those from CPD samples if TOTO processing is applied beforehand. One particular advantage of freeze-drying, though, is that after freezing, brittle fracture through the tissue can occur making examination of intracellular structure by SEM relatively easy. However, again, TOTO processing prior to freezing is of value as this appears to prevent the formation of large ice-crystal during freezing. Examination of isolated outer hair cells by SEM shows that isolation procedures do not cause significant damage to the stereociliary bundles.

First appearance and development of motile properties in outer hair cells isolated from guinea-pig cochlea.

Cochleae from fetal guinea-pigs (37 to 64 gestation days, gd) were used to correlate the appearance of motile properties of isolated outer hair cells (OHCs) with the development of specific morphological features. Both the 'fast' electrically-driven and the 'slow' calcium-induced motilities appeared first in OHCs from basal turn of 52 gd fetuses. At 56 gd, most of basal and some apical OHCs responded positively to both types of stimulation. All tested cells were positive at 64 gd. It is noteworthy that this period closely corresponds to the onset and maturation of the gross cochlear potentials. Some structural changes in the organ of Corti may be correlated with the development of OHC motile properties: the acquisition of an adult-like cylindrical shape by the OHC, its lateral detachment from neighboring Deiters cells, and its surrounding by fluid spaces. At the ultrastructural level, the formation of a first layer of laminated cisternae regularly aligned along the OHC plasma membrane from the cuticular plate down to the nuclear level, temporally coincided with the onset of in vitro motility (52 gd). The following days, pillars and a sub-membrane lattice were clearly noticed between the outermost cisternal membrane and the plasma membrane. The results support the ideas that: motile properties observed in vitro reflect the in vivo active mechanisms, and that one single layer of laminated cisternae and its associated sub-plasma membrane material may be needed for OHC motility.

Assessment of ultrastructure in isolated cochlear hair cells using a procedure for rapid freezing before freeze-fracture and deep-etching.

Separated cochlear outer hair cells and isolated strips of organ of Corti containing hair cells and supporting cells have been rapidly frozen before freeze-fracture and deep-etching by immersion of samples sandwiched between two copper plates into liquid nitrogen-cooled propane: isopentane. Assessment of this procedure has shown that no significant freezing damage occurs. The ultrastructure of the hair cells revealed by freeze-fracture of these non-chemically fixed preparations was generally very similar to that seen in fixed material. This indicates that the processing of cochlear tissue normally used for electron microscopy produces few obvious structural artefacts. It also demonstrated that procedures for isolating cochlear hair cells generally do not affect cell structure significantly. However, some isolated hair cells did show abnormalities within the membranes of the lateral cisternae. Such membrane alterations, which would not be identified by light microscopy, occurred to a variable extent but were more commonly present after prolonged periods in maintenance medium. Deep-etching of the preparations to examine extracellular features around stereocilia revealed clearly lateral cross-links between stereocilia. However, tip-links could not be positively identified in either unfixed or prefixed preparations.

Shape changes in isolated outer hair cells: measurements with attached microspheres.

Shape changes can be induced in isolated outer hair cells by various stimuli and quantified from digitized video-images. While overall changes in length between base and apex are easily measured, changes in defined segments of the cell require fixed landmarks on the cell body. The problem of locating such landmarks makes it difficult to assess if a change in length is uniform or largely confined to a particular segment of the cell. This information is important in identifying the location of a contractile apparatus and the elucidation of mechanisms of motility. We demonstrate here that microspheres can serve as reference points for such measurements. By attaching microspheres to cells we determined that, when outer hair cells increased their volume upon K(+)-depolarization, their middle segment shortened more significantly (14 +/- 6%) than either the basal (10 +/- 5%) or apical section (7 +/- 6%; P less than 0.01). In contrast, when cortical contractions were induced by elevating intracellular Ca2+, the elongation of the cells was more pronounced in their basal (8 +/- 2%) than their apical (6 +/- 2%; P = 0.06) or middle region (6 +/- 3%). This study provides further insight into the mechanisms of shape changes in isolated outer hair cells and illustrates a method to analyze localized changes in the absence of internal landmarks.

Differential motile response of isolated inner and outer hair cells to stimulation by potassium and calcium ions.

Inner and outer hair cells were mechanically isolated from the guinea pig cochlea and subjected to stimuli known to induce shape changes in outer hair cells. Depolarization by 70 mM KCl which causes osmotic swelling of outer hair cells also swelled inner hair cells by approximately 8% of their volume. The application of the calcium ionophore ionomycin which induces cortical contractions and elongation of outer hair cells, did not affect the shape of inner hair cells. Since ionomycin increased free intracellular calcium levels in both inner and outer hair cells, the results demonstrate that inner hair cells do not possess the mechanisms necessary for a contractile response to calcium. Thus, calcium is a specific regulator of outer hair cell motility making this mechanism a likely physiological modulator of a transduction feedback process.

Monoclonal antibodies to inner ear antigens: I. Antigens expressed by supporting cells of the guinea pig cochlea.

Murine monoclonal antibodies against guinea pig cochlear epithelium were generated with the goal of identifying cochlea-specific antigens and elucidating their function. To compensate for the limited amount of cochlear tissue, intrasplenic immunization was used. Hybridoma supernatants were screened by ELISA for antibody production and for binding to homogenates from cochlea, liver, lung, kidney and brain. Hybrids producing antibody to cochlea were subcloned and tested immunocytochemically against frozen sections and surface preparations of paraformaldehyde-fixed cochlear tissue. KHRI-1, a low titer IgM antibody stained only Hensen cells. KHRI-2, also an IgM antibody, stained tectorial membrane, cells of the spiral limbus, cells bordering the space of Nuel, Hensen cells and the root cells of the spiral prominence. KHRI-3, an IgG1 antibody, stained the phalangeal processes of outer pillar cells and the apical portion of phalangeal processes of Deiters' cells in a distinctive wine goblet pattern on surface preparations. KHRI-3 antibody also reacted with peripheral nerves and pia mater of brain in unfixed frozen sections but the antigenic site was not stable to fixation in contrast to the epitope detected in the cochlea. In Western blots of detergent extracts from cochlea KHRI-3 stained a broad tissue-specific band of Mr 70-75 kDa; a narrower band of Mr 68-70 kDa was identified by KHRI-3 in extracts of tongue and brain. KHRI-1 and KHRI-2 did not detect any proteins in Western blots. The monoclonal antibodies KHRI-1, -2, and -3 which define epitopes expressed by discrete populations of supporting cells in the inner ear should be useful in characterizing the nature and function of cellular structures in the cochlea.

Acetylcholine, carbachol, and GABA induce no detectable change in the length of isolated outer hair cells.

The mechanical and electrical properties of cochlear outer hair cells (OHCs) are suggested to modulate transduction by inner hair cells. These properties of OHCs are presumably regulated by efferent neurons which use several transmitters including acetylcholine (Ach) and gamma aminobutyric acid (GABA). Since it had been suggested that Ach causes isolated OHCs to shorten visibly, this study was designed to investigate whether GABA also alters the length of OHCs. OHCs were isolated from the guinea pig cochlea by mechanical dispersion after collagenase treatment. Cells were initially selected by strict morphological criteria. In addition they were only included in further studies if they attained a constant length during 10 min of superfusion with buffer solution. Neither GABA (20 microM: 100 microM), Ach (5 mM; 10 microM with 10 microM eserine) or carbachol (10 microM; 100 microM) altered OHC length when applied in iso-osmotic Hank's balanced salt solution (total number of cells tested, 72). If a change in length occurred it must have been smaller than 0.3 microns, our detection ability. In contrast, high potassium and variations in osmolarity changed hair cell length by 3-10% in agreement with other reports.

Increasing intracellular free calcium induces circumferential contractions in isolated cochlear outer hair cells.

The relationship between intracellular free calcium and the motile responses of outer hair cells isolated from the guinea pig cochlea was examined. Calcium levels were modulated by the addition of the calcium ionophores ionomycin or A23187 to the incubation medium and monitored with the fluorescent calcium indicator fluo-3. In the presence of 1.25 mM external calcium, the application of either ionophore (10 microM) led to an increase in intracellular free calcium from 157 +/- 76 nM to 1200 +/- 500 nM within 30-60 sec. Concurrently, cells elongated by 1-2 microns, cell diameter decreased, and cell volume shrank by 269 +/- 220 microns 3 (5.0 +/- 4.1%). The reduction in diameter was most pronounced in the middle portion of the cell (4.4% +/- 4.2%), also evident in the apical region (3.1% +/- 4.8%) but not significant in the basal region near the nucleus. This response was observed in outer hair cells from basal and apical turns of the cochlea and was reversed when the cells were rinsed with calcium-free medium supplemented with 2 mM EGTA. Optical imaging of the cell membrane with the potentiometric dye 1-(3-sulfonatopropyl)-4-[beta] [2-(di-n-butylaminol)-6-naphthyl vinyl] pyridinium betaine during the elevation of intracellular calcium demonstrated features of contractility at the lateral cell membrane. A rise in intracellular calcium as well as the motile response was still observed after a 5-min exposure of the cells to a calcium-free solution (supplemented with 2 mM EGTA), indicating that the ionophore was also able to liberate calcium from intracellular sites. However, depletion of calcium stores through prolonged incubation of the cells in calcium-free medium (30-60 min) suppressed both the calcium signal and the cell response. The calmodulin inhibitors trifluoperazine and pimozide (30 microM) blocked the cell motility induced by ionomycin while they left the increase of intracellular calcium unaffected. These observations suggest that calcium-dependent circumferential contractions in outer hair cells are mediated by calmodulin. The application to the extracellular medium of putative neurotransmitters of the cochlear efferent system such as acetylcholine and GABA led to neither an increase in intracellular calcium nor a modification of cell shape. Therefore, these neurotransmitters may not be directly involved in calcium-induced contractions in outer hair cells. The circumferential contractions altered the stiffness of the plasma membrane and the turgor of the cell. Under normal conditions, changes in cell volume were inversely proportional to the osmotic pressure of the extracellular medium following van't Hoff's law.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells.

Cochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short-term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization-induced transmembrane calcium flux, and depolarization-induced motile responses. On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K+]-depolarization, intracellular calcium levels (monitored with the fluorescent calcium-sensitive dye fluo-3) increased from a resting value of 218 +/- 102 nM to 2,018 +/- 1,077 nM concomitant with a cell shortening of 0.7% +/- 1.3%. The depolarization-induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium-channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K+]-induced calcium increase at an IC50 of 50 microM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K+]-depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% +/- 1.4%). This argues effectively against the existence of a calcium-dependent actomyosin-mediated component in [K+]-induced shape changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Photo-induced irreversible shortening and swelling of isolated cochlear outer hair cells.

Living outer hair cells were irradiated under conditions of fluorescent microscopy (epi-illumination through the objective) with UVR (waveband 340-380 nm), blue light (waveband 450-490 nm) or green light (waveband 515-560 nm) in the intracellular presence or absence of the fluorescent dyes fura-2 or 2',7'-bis-(carboxyethyl) 5-(and 6-)carboxyfluorescein (BCECF). In response to UVR with or without intracellular fura-2 and to blue light in the presence of BCECF (irradiation intensities of 2-12 x 10(5) W/m2), the cells shortened and swelled within 15-30 s, accompanied by the formation of numerous cytoplasmic granulations. The cellular reactions were significantly delayed to 3 min by the addition of 1 mM of the radical scavengers p-phenylenediamine or n-propyl gallate. This protection suggests that free radicals, produced under UVR or under blue light irradiation in the presence of the sensitizer BCECF, are possible causative agents of this cell damage. The response of the photo-damaged cells, namely shortening and increase in volume, resembled the characteristics of hair cells exposed to an hypo-osmotic shock. This suggests that structural alterations of the cytoplasmic membrane and the sub-membrane cortex occurred under photo-irradiation, and that these structures can be implicated in the maintenance of the elongated cylindrical shape of the outer hair cells, possibly by maintaining intracellular hyperosmolarity.

Gentamicin alters membrane structure as shown by freeze-fracture of liposomes.

Freeze-fracture has been used to examine the effects of gentamicin on membrane structure in liposomes of different anionic phospholipids combined with a neutral phospholipid, phosphatidylcholine. The molar ratios of neutral: anionic lipid were 1:1 (high anionic lipid ratio) and 4:1 (low anionic lipid) and the liposomes were incubated with 0.1 mM (low) and 1 mM (high) gentamicin. With the anionic phospholipid phosphatidylinositol bisphosphate, an identifiable disruption of the membrane bilayer was observed as well as aggregation of liposomes leading to membrane fusion. These effects occurred both at low gentamicin concentration and low anionic lipid content of the liposomes; these responses were not inhibited by 1 mM Ca2+. With the other anionic lipids tested (phosphatidylserine, phosphatidylinositol and phosphatidylinositol monophosphate), only aggregation and fusion of liposomes was observed and this effect only occurred at high gentamicin concentration and high anionic lipid content. Further, 1 mM Ca2+ inhibited the responses of these other anionic lipids to gentamicin. The results demonstrate the unique character of the interaction between gentamicin and phosphatidylinositol bisphosphate and provide further support for the hypothesis that a specific binding to this lipid is a key step in the ototoxic action of aminoglycoside antibiotics. They also suggest that such an interaction in vivo might cause alterations to the structure and properties of cell membranes in the inner ear.

Characteristics of the membrane of the stereocilia and cell apex in cochlear hair cells.

Freeze-fracture has been used to examine the membrane of the cell apex and of the stereocilia in cochlear hair cells. The apical (non-stereociliary) membrane of inner hair cells (IHCs) exhibited a lower density of intramembrane particles (IMP) than that of the outer hair cells (OHCs) but in both cell types the apical membrane responded to the effects of filipin. The distribution of IMP and of filipin-induced membrane deformations was uniform over the apical membranes in both IHC and OHC, thus, providing no evidence for local membrane differentiation on the non-stereociliary part of the hair cell apex. The stereociliary membranes of IHC and of OHC differed not only in the density of IMP, but also in their responses to filipin and to tomatin. IHC stereocilia responded intensely to both agents. OHC stereocilia showed a significantly lower density of filipin-induced lesions and appeared almost unaffected by tomatin. This suggests that the OHC stereocilial membrane may be structurally specialized. The membrane at the apical end of stereocilia appeared to be differentiated from the membrane of the stereociliary shaft. The tip region was free of the usual IMP and showed no filipin-induced lesions. The differentiation at the apical end was also apparent in samples which have been rapidly frozen without prior chemical fixation or cryoprotection, showing that the particle-free area was not an artefact induced by glutaraldehyde fixation. Close examination of the membrane at the apical-most tip of the stereocilium revealed the presence of a small number of large particles of 10.5-11.0 nm diameter. The occurrence of membrane differentiation localized to the tip of the stereocilium may be consistent with the suggestion that transduction channels in hair cells are situated at this point.

Species differences in the distribution of infracuticular F-actin in outer hair cells of the cochlea.

The distribution of filamentous actin (F-actin) in outer hair cells has been examined in several mammalian species using tetramethylrhodamine phalloidin, a specific marker for F-actin. The stereocilia and cuticular plates of the OHC in all species examined (pigmented guinea pig, hooded rat, chinchilla and squirrel monkey) contained F-actin; however, an infracuticular network of F-actin was present in OHC of the apical turns of the guinea pig cochlea but could not be identified in any other species examined.

Differences in the distribution of F-actin in outer hair cells along the organ of Corti.

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.