Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.     

Cytoplasmic actin and cochlear outer hair cell motility

Summary Isolated outer hair cells of the guinea pig lacking a cuticular plate and its associated infracuticular network retain the ability to shorten longitudinally and become thinner. Membrane ghosts lacking cytoplasm retain the cylindrical shape of the hair-cell, and although they do not shorten, they retain the ability to constrict and become thinner. These data suggest that cytoplasmic components are associated with outer hair-cell longitudinal shortening and that the lateral wall is responsible for maintaing cell shape and for constriction. Actin, a protein associated with the cytoskeleton and cell motility, is thought to be involved in outer hair-cell motility. To study its role, actin was localized in isolated outer hair cells by use of phalloidin labeled with fluorescein and antibodies against actin coupled to colloidal gold. In permeabilized guinea-pig hair cells stained with phalloidin, actin filaments are found along the lateral wall. In frozen-fixed hair cells actin filaments are distributed uniformly throughout the cytoplasm. Electron-microscopic studies show that antibodies label actin throughout the outer hair-cell body. Thus cytoplasmic actin filaments may provide the structural basis for the contraction-like events.     

Concanavalin A protects hair cells against gentamicin ototoxicity in rat cochlear explant cultures

Degeneration of hair cells and spiral ganglion neurons due to acoustic trauma and various ototoxins is a major cause of hearing loss. Although our previous study demonstrates that specific neurotrophins protect spiral ganglion neurons from ototoxic insult, they do not protect hair cells. In the present experiments, we used postnatal rat cochlear explant cultures to identify molecules that protect hair cells from ototoxic damage. Of 51 compounds examined, only concanavalin A (Con A) significantly protected hair cells from gentamicin. A dose-dependent study of Con A showed that maximal protection occurred at 100 nM. The protective effects of Con A on hair cells were confirmed with confocal microscopy and paraffin sectioning of the cultures. Several experiments were performed to examine the mechanism of protection by Con A. Incubation of Con A with gentamicin did not form a complex and coaddition of Con A and gentamicin to Escherichia coli cultures did not interfere with the antibiotic activity of gentamicin. However, Lyso-Tracker staining and gentamicin immunocytochemistry provided evidence that preincubation with Con A blocked gentamicin uptake into hair cells. Considered together, these findings may help elucidate the ototoxic mechanism of aminoglycoside antibiotics, and suggest that Con A may be of therapeutic value in prevention of aminoglycoside-induced hearing loss.     

ROCK-dependent and ROCK-independent control of cochlear outer hair cell electromotility

Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000-28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier.     

Simulation of motor-driven cochlear outer hair cell electromotility.

We propose a three-dimensional (3D) model to simulate outer hair cell electromotility. In our model, the major components of the composite cell wall are explicitly represented. We simulate the activity of the particles/motor complexes in the plasma membrane by generating active strains inside them and compute the overall response of the cell. We also consider the constrained wall and compute the generated active force. We estimate the parameters of our model by matching the predicted longitudinal and circumferential electromotile strains with those observed in the microchamber experiment. In addition, we match the earlier estimated values of the active force and cell wall stiffness. The computed electromotile strains in the plasma membrane and other components of the wall are in agreement with experimental observations in trypsinized cells and in nonmotile cells transfected with Prestin. We discover several features of the 3D mechanism of outer hair cell electromotilty. Because of the constraints under which the motors operate, the motor-related strains have to be 2-3 times larger than the observable strains. The motor density has a strong effect on the electromotile strain. Such effect on the active force is significantly lower because of the interplay between the active and passive properties of the cell wall.     

Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In this system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Hsp70 overexpression inhibits aminoglycoside-induced hair cell death in vitro. In this study, we utilized Hsp70-overexpressing mice to determine whether Hsp70 is protective in vivo. Both Hsp70-overexpressing mice and their wild-type littermates were treated with systemic kanamycin (700 mg/kg body weight) twice daily for 14 days. While kanamycin treatment resulted in significant hearing loss and hair cell death in wild-type mice, Hsp70-overexpressing mice were significantly protected against aminoglycoside-induced hearing loss and hair cell death. These data indicate that Hsp70 is protective against aminoglycoside-induced ototoxicity in vivo.     

Reorganization of cytoskeletal and junctional proteins during cochlear hair cell degeneration.

Experiments were carried out to elucidate changes in cytoskeletal elements and intercellular junctions in the organ of Corti, when hair cells degenerate and phalangeal scars form. Hair cell damage was induced by exposing guinea pigs to high intensity noise. The spatial and temporal changes in the organization of microfilaments, intermediate filaments, and tight junction-specific proteins were investigated using scanning and transmission electron microscopy and histochemistry. The results show that microfilaments, cytokeratins, adherens junctions, and tight junctions rearrange their distribution in damaged areas. From the temporal sequence of these changes it appears that phalangeal scars develop simultaneous with hair cell degeneration, and that the integrity of the luminal membranes in the organ of Corti is not interrupted. Each scar is formed by two supporting cells which expand and invade the sub-apical region of the dying hair cell. This region becomes cytokeratin-positive. The two supporting cells meet at the mid-line of the scar, where a new junctional complex is formed. The junctional complex consists of tight junction and adherens-type junction, but desmosomes are absent.     

Regulation of p27Kip1 during gentamicin mediated hair cell death

The INK4 and Kip/Cip families of Cyclin Dependent Kinase inhibitors (CKIs) are regulators of the cell cycle. In addition, CKIS including p27^Kip1can protect cells from apoptosis in vitro. However, little is known about protective effect of p27^Kip1in vivo. We used systemic treatment with aminoglycosides to induce hair-cell death in the basilar papilla (BP), the auditory organ of the avian inner ear, and characterised the expression of p27^Kip1with confocal and immunofluorescence microscopy. In contrast to the adult mammalian cochlea where p27^Kip1is expressed only in supporting cells, p27^Kip1is found in the nuclei of both hair cells and supporting cells in the BP of the normal, mature bird. Forty-eight hours after gentamicin treatment, hair cells with TUNEL positive nuclei and hair cells with pyknotic nuclei were both detected, suggesting many hair cells die by apoptosis. When the BP was double labelled for p27^Kip1and myosin VIIa, a hair-cell specific protein, all dying hair cells that had been ejected from the epithelium were found to be myosin VIIa positive but negative for p27^Kip1even though nuclear remnants were still visible. In the transition zone where partial hair-cell loss occurs, freshly ejected hair cells lying immediately above the surface of the BP no longer expressed p27^Kip1. Damaged hair cells within the epithelium in the transition zone contained p27^Kip1in their cytoplasm but not in their nuclei. These data support recent in vitro findings suggesting that p27^Kip1protects cells from apoptosis and that its downregulation may be a general feature of programmed cell death.     

The role of outer hair cell motility in cochlear tuning.

The mammalian cochlea's remarkable sensitivity and frequency selectivity are thought to be mediated by the mechanical feedback action of outer hair cells. New tools for measuring the movement of cochlear elements, and recent advances in modeling are increasing our knowledge of cochlear mechanics.     

Dynamic changes in hair cell stereocilia and cochlear transduction after noise exposure

The structures of cochlear transduction include stereocilia at the apical surface of hair cells and their connection to the tectorial membrane. The transduction site is one of the loci for noise-induced cochlear damage. Although stereocilia are susceptible to noise, it has been found that in the inner ears of avians, this fragile structure is largely self-repairing and is associated with recovery of hearing sensitivity after noise exposure, as observed in the difference between the temporal threshold shift (TTS) and the permanent threshold shift (PTS). In the mammalian cochleae, however, threshold shifts measured in the auditory brainstem responses (ABR) did not parallel the chronological changes in the stereocilia on hair cells. It is unclear how the morphological recovery of the stereocilia on the mammalian hair cells is correlated with the changes in cochlear transduction that can be assessed by measuring receptor potential. In the present study, guinea pigs were exposed to a broadband noise of 110 dB SPL for 2 h. Auditory sensitivity was evaluated using ABR and cochlear transduction was assessed using cochlear microphonics (CM). Stereocilia morphology was quantified at different time points after the noise and compared with the control. The noise produced a TTS of 55.69 ± 14.13 dB in frequency-averaged ABR thresholds. The threshold shift was reduced to 9.58 ± 11.75 dB SPL 1 month later with virtually no loss of hair cells. Damage to the stereocilia immediately after noise exposure was found to be associated with depression of CM amplitude. Virtually no abnormal stereocilia were observed 1 month after the noise in association with a fully recovered CM.     

Modes and balance of energy in the piezoelectric cochlear outer hair cell wall

Here, we analyze energy transformations in the outer hair cell and its effectiveness as a piezoelectric-type actuator in the cochlea. The major modes of energy are introduced, and a method to estimate the coefficients of their tension-dependence is proposed. Next, we derive balance of the mechanical and electrical parts of energy, and show two forms of the active energy associated with the motors driving electromotility. The two forms of the active energy, stored mechanical energy, and external electrical work are then introduced as functions of voltage and applied force. We use the energy balance to introduce and estimate the effectiveness of the cell's electromotile response.     

Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification

It is a central tenet of cochlear neurobiology that mammalian ears rely on a local, mechanical amplification process for their high sensitivity and sharp frequency selectivity. While it is generally agreed that outer hair cells provide the amplification, two mechanisms have been proposed: stereociliary motility and somatic motility. The latter is driven by the motor protein prestin. Electrophysiological phenotyping of a prestin knockout mouse intimated that somatic motility is the amplifier. However, outer hair cells of knockout mice have significantly altered mechanical properties, making this mouse model unsatisfactory. Here, we study a mouse model without alteration to outer hair cell and organ of Corti mechanics or to mechanoelectric transduction, but with diminished prestin function. These animals have knockout-like behavior, demonstrating that prestin-based electromotility is required for cochlear amplification.     

Model of cochlear microphonic explores the tuning and magnitude of hair cell transduction current

The mammalian cochlea relies on the active forcing of sensory outer hair cells (OHCs) to amplify traveling wave responses along the basilar membrane. These forces are the result of electromotility, wherein current through the OHCs leads to conformational changes in the cells that provide stresses on surrounding structures. OHC transducer current can be detected via the voltage in the scala tympani (the cochlear microphonic, CM), and the CM can be used as an indicator of healthy cochlear operation. The CM represents a summation of OHC currents (the inner hair cell contribution is known to be small) and to use CM to probe the properties of OHC transduction requires a model that simulates that summation. We developed a finite element model for that purpose. The pattern of current generators (the model input) was initially based on basilar membrane displacement, with the current size based on in vitro data. The model was able to reproduce the amplitude of experimental CM results reasonably well when the input tuning was enhanced slightly (peak increased by ∼6 dB), which can be regarded as additional hair bundle tuning, and with a current/input value of 200–260 pA/nm, which is ∼4 times greater than the largest in vitro measures.     

Discharge patterns of chicken cochlear ganglion neurons following kanamycin-induced hair cell loss and regeneration

Hair cells in the basal, high frequency region (>1100 Hz) of the chicken cochlea were destroyed with kanamycin (400 mg/kg/d × 10 d) and allowed to regenerate. Afterwards, single unit recordings were made from cochlear ganglion neurons at various times post-treatment. During the first few weeks post-treatment, only neurons with low characteristic frequencies (<1100 Hz) responded to sound. Despite the fact that the low frequency region of the cochlea was not destroyed, neurons with low characteristic frequencies had elevated thresholds, abnormally broad U-shaped or W-shaped tuning curves and low spontaneous discharge rates. At 2 days post-treatment, the spontaneous discharge rates of some acoustically unresponsive units fluctuated in a rhythmical manner. As recovery time increased, thresholds decreased, tuning curves narrowed and developed a symmetrical V-shape, spontaneous rate increased and neurons with higher characteristic frequencies began to respond to sound. In addition, the proportion of interspike interval histograms with regularly spaced peaks increased. These improvements progressed along a low-to-high characteristic frequency gradient. By 10–20 weeks post-treatment, the thresholds and tuning curves of neurons with characteristic frequencies below 2000 Hz were within normal limits; however, the spontaneous discharge rates of the neurons were still significantly lower than those from normal animals.Abbreviations KM kanamycin - BrdU bromodeoxyuridine - CF characteristic frequency - CAP compound action potential - ISI interspike interval     

Calcium dependence of exocytosis and endocytosis at the cochlear inner hair cell afferent synapse

Release of neurotransmitter at the inner hair cell (IHC) afferent synapse is a fundamental step in translating sound into auditory nerve excitation. To study the Ca2+ dependence of the underlying vesicle fusion and subsequent endocytosis, we combined Ca2+ uncaging with membrane capacitance measurements in mouse IHCs. Rapid elevations in [Ca2+]i above 8 microM caused a biphasic capacitance increase corresponding to the fusion of approximately 40,000 vesicles. The kinetics of exocytosis displayed a fifth-order Ca2+ dependence reaching maximal rates of >3 x 10(7) vesicle/s. Exocytosis was always followed by slow, compensatory endocytosis (tau congruent with 15 s). Higher [Ca2+]i increased the contribution of a faster mode of endocytosis with a Ca2+ independent time constant of approximately 300 ms. These properties provide for rapid and sustained transmitter release from this large presynaptic terminal.     

Adaptive evolution in mammalian proteins involved in cochlear outer hair cell electromotility

Somatic electromotility in cochlear outer hair cells, as the basis for cochlear amplification, is a mammalian novelty and it is largely dependent upon rapid cell length changes proposed to be mediated by the motor-protein prestin, a member of the solute carrier anion-transport family 26. Thus, one might predict that prestin has specifically evolved in mammals to support this unique mammalian adaptation. Using codon-based likelihood models we found evidences for positive selection in the motor-protein prestin only in the mammalian lineage, supporting the hypothesis that lineage-specific adaptation-driven molecular changes endowed prestin with the ability to mediate somatic electromotility. Moreover, signatures of positive selection were found on the alpha10, but not the alpha9, nicotinic cholinergic receptor subunits. An alpha9alpha10-containing nicotinic cholinergic receptor mediates inhibitory olivocochlear efferent effects on hair cells across vertebrates. Our results suggest that evolution-driven modifications of the alpha10 subunit probably allowed the alpha9alpha10 heteromeric receptor to serve a differential function in the mammalian cochlea. Thus, we describe for the first time at the molecular level signatures of adaptive evolution in two outer hair cell proteins only in the lineage leading to mammals. This finding is most likely related with the roles these proteins play in somatic electromotility and/or its fine tuning.