Direct administration of 2-Hydroxypropyl-Beta-Cyclodextrin into guinea pig cochleae: Effects on physiological and histological measurements.

2-Hydroxypropyl-Beta-Cyclodextrin (HPßCD) can be used to treat Niemann-Pick type C disease, Alzheimer's disease, and atherosclerosis. But, a consequence is that HPßCD can cause hearing loss. HPßCD was recently found to be toxic to outer hair cells (OHCs) in the organ of Corti. Previous studies on the chronic effects of in vivo HPßCD toxicity did not know the intra-cochlear concentration of HPßCD and attributed variable effects on OHCs to indirect drug delivery to the cochlea. We studied the acute effects of known HPßCD concentrations administered directly into intact guinea pig cochleae. Our novel approach injected solutions through pipette sealed into scala tympani in the cochlear apex. Solutions were driven along the length of the cochlear spiral toward the cochlear aqueduct in the base. This method ensured that therapeutic levels were achieved throughout the cochlea, including those regions tuned to mid to low frequencies and code speech vowels and background noise. A wide variety of measurements were made. Results were compared to measurements from ears treated with the HPßCD analog methyl-ß-cyclodextrin (MßCD), salicylate that is well known to attenuate the gain of the cochlear amplifier, and injection of artificial perilymph alone (controls). Histological data showed that OHCs appeared normal after treatment with a low dose of HPßCD, and physiological data was consistent with attenuation of cochlear amplifier gain and disruption of non-linearity associated with transferring acoustic sound into neural excitation, an origin of distortion products that are commonly used to objectively assess hearing and hearing loss. A high dose of HPßCD caused sporadic OHC losses and markedly affected all physiologic measurements. MßCD caused virulent destruction of OHCs and physiologic responses. Toxicity of HPßCD to OHC along the cochlear length is variable even when a known intra-cochlear concentration is administered, at least for the duration of our acute studies.

BK Channels in the Vertebrate Inner Ear.

The perception of complex acoustic stimuli begins with the deconstruction of sound into its frequency components. This spectral processing occurs first and foremost in the inner ear. In vertebrates, two very different strategies of frequency analysis have evolved. In nonmammalian vertebrates, the sensory hair cells of the inner ear are intrinsically electrically tuned to a narrow band of acoustic frequencies. This electrical tuning relies on the interplay between BK channels and voltage-gated calcium channels. Systematic variations in BK channel density and kinetics establish a gradient in electrical resonance that enables the coding of a broad range of acoustic frequencies. In contrast, mammalian hair cells are extrinsically tuned by mechanical properties of the cochlear duct. Even so, mammalian hair cells also express BK channels. These BK channels play critical roles in various aspects of mammalian auditory signaling, from developmental maturation to protection against acoustic trauma. This review summarizes the anatomical localization, biophysical properties, and functional contributions of BK channels in vertebrate inner ears. Areas of future research, based on an updated understanding of the biology of both BK channels and the inner ear, are also highlighted. Investigation of BK channels in the inner ear continues to provide fertile research grounds for examining both BK channel biophysics and the molecular mechanisms underlying signal processing in the auditory periphery.

Tamoxifen alters gating of the BK alpha subunit and mediates enhanced interactions with the avian beta subunit.

Mammalian BK channels are modulated by estrogen and non-steroidal estrogen-like compounds (i.e. xenoestrogens), but the effects are dependent on channel composition. (Xeno)estrogens preferentially activate BK channels through accessory beta subunits, but reduce single-channel conductance by interaction with alpha subunits. In this report, the xenoestrogen tamoxifen was applied to chicken BK channels, in order to asses the mechanism behind drug interaction and to determine the extent to which (xeno)estrogen interaction is extended to avian BK homologs. As with mammalian isoforms, the properties of chicken BK channels were modulated by tamoxifen in a subunit-dependent manner. Tamoxifen reduced single-channel conductance through interaction with the alpha subunit. However, if the expression construct included the beta subunit, tamoxifen increased the channel's open probability and shifted the voltage-activation range to more negative potentials. This effect on channel gating was concentration-dependent, with an EC(50) of about 0.2 microM. Tamoxifen-mediated reductions in gating charge and in the intrinsic energetics that govern channel equilibrium. The relative contribution of these two effects on channel gating was altered by beta co-expression. Modulation by (xeno)estrogens may be an evolutionarily conserved mechanism for non-genomic hormonal actions, and the limited conservation between avian and mammalian beta subunits may suggest potential binding motifs. Alternatively, the data are consistent with a tamoxifen-mediated conformation change in the alpha subunit that alters the way alpha and beta subunits interact, resulting in enhanced gating without direct binding to beta.

A finite-element model of inner ear hair bundle micromechanics.

Understanding hair-cell micromechanics is central to the discussion of mechanotransduction in these cells. This paper presents a finite-element model that characterizes the stiffness and deflection properties of an inner-ear hair bundle. Average morphological dimensions were used for sterocilia height (6, 8, and 10 microns), diameter (0.25 microns), and rootlet separation (0.5 microns) for a single bundle column containing three rows. Stereocilia material properties were described as isotropic, homogeneous, linearly elastic, and nearly incompressible. Young's modulus for the stereocilia ranged from a maximum of actin and down. The column of stereocilia were coupled by linear elastic material modeling tip and lateral links. When the hairs were deflected by a static force applied to the tip of the tallest cilium, the hair-bundle model yielded a stiffness of 9.5 x 10(-4) to 21 x 10(-4) N/m, which was in the range of typical experimental values but approximately a factor of 4-10 times the average of all experimental values. Model parameters such as bundle size, shape, and material properties were systematically varied to determine each component's contribution to bundle stiffness. Additionally, tip-link tensions were determined for a range of deflections in a five cilium model and were shown to be proportionally graded in magnitude along the bundle staircase.

Low calcium abolishes tip links and alters relative stereocilia motion in chick cochlear hair cells.

The role of stereocilia tip links in controlling hair bundle motion on chick hair cells was examined in this study. Hair cells from the apical end of the basilar papilla were maintained in culture medium and oriented so that the sensory hair bundles were viewed in profile. A water-jet was used to stimulate the hair bundle and stroboscopic illumination allowed slow motion viewing of a sensory hair motion at the bundle edges. Motion of the tallest stereocilium in the bundle was set to a criterion angular deflection and the excursion of the shortest stereocilium was measured. These measurements were made in a sample of hair cells maintained in culture medium containing either near normal levels of calcium or very low calcium levels supplemented with EGTA. In low calcium the angular deflection of the shortest hair was significantly reduced from that observed in normal media. The resting inward tilt of the hairs in the bundle, however, did not change. Scanning electron microscopy verified an almost complete destruction of tip links after exposure to low calcium. These results suggest that tip links contribute significantly to the relative motion of stereocilia and exhibit the mechanical properties of a relatively stiff linkage.

The tip link's role in asymmetric stereocilia motion of chick cochlear hair cells.

The symmetry of chick cochlear hair bundle motion was examined in this study. Isolated segments from the basilar papilla were incubated in vitro in either normal or low calcium medium, which is known to disrupt tip links. Stereociliary bundles, stimulated with an oscillating water microjet, were oriented in profile and viewed in slow motion at high magnification with stroboscopic illumination. The displacement of the tallest hair in the bundle was fixed to 20 degrees peak-to-peak (P-P) motion. The angular deflections of the shortest and tallest hairs were then measured in both the positive (towards the tallest hair) and negative (towards the shortest) directions with respect to the non-stimulated position of the hair. The tallest hairs exhibited nearly symmetric motion in medium containing normal and low calcium. The shortest hairs, in normal calcium, displayed considerable asymmetry with angular deflections in the positive direction significantly larger than in the negative direction. This asymmetric motion disappeared after incubation in low calcium. The shortest hair angular displacement in the negative direction, however, was the same in both normal and low calcium conditions. These results indicated that the tallest and shortest hairs moved with equal angular deflection in the negative direction, while in the positive direction the shortest hair moved through a significantly greater angular deflection than the tallest hair. The implication of this finding is that the tip links contributed significantly to hair bundle motion in the positive direction only.

Distal separation of chick cochlear hair cell stereocilia: analysis of contact-constraint models.

One model often used in the study of hair bundle micromechanics assumes simple geometric relationships between hair displacements, constrained by contact between neighboring hairs at their distal tips. Recent observations of hair bundle motion provided the opportunity to evaluate the contact-constraint model against measured displacements for the tallest and shortest sensory hairs. A contact-constraint model was developed based on the geometry of a single column of stereocilia. The model used morphological data from chick hair bundles for which displacement data in the excitatory and inhibitory directions were also available. For each hair bundle, a unique sensory hair radius was determined so that the calculated resting bundle morphology matched the measured values. The model was then evaluated against the displacement data for each hair bundle. In each case, the model underestimated the excitatory displacement of the shortest hairs. Failure of the model to accurately predict bundle motion raises the possibility of a distal separation between the hairs at rest. It is suggested that tip links pull the hairs through this separation during excitatory deflections. Perhaps at damaging levels of displacement, the hairs suddenly come into contact, tip-link tension dramatically increases, and the tip-links are fractured.

P2X antagonists inhibit styryl dye entry into hair cells.

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 microM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 microM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 microM). At a concentration that also blocks P2X(2) receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.

Cloning and characterization of SK2 channel from chicken short hair cells.

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74+/-0.17 microM. The expressed channels were blocked by apamin (IC(50)=73.3+/-5.0 pM) and d-tubocurarine (IC(50)=7.6+/-1.0 microM), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.

Stereocilium injury mediates hair bundle stiffness loss and recovery following intense water-jet stimulation.

Inner ear hair cells exhibit many pathologies following exposure to intense sound, and the hair bundle is a major site of damage. This paper measures in vitro hair bundle motion on chick cochlear hair cells after intense in vitro and in vivo stimulation to explore the nature of hair bundle injury. Hair bundle stiffness, as well as relative and asymmetric motion of individual stereocilia, is controlled largely by the extracellular tip links, and a change in hair bundle motion was used to assess tip-link destruction following overstimulation. Intense in vitro stimulation caused a loss in stiffness that fully recovered within 10 min post-exposure. Relative and asymmetric stereocilia motion, however, were unchanged following the exposure, implying that tip links remained intact while the core or rootlet of the stereocilia were damaged and subsequently repaired. Intense and prolonged in vivo sound exposures produced stereocilia movements, measured in vitro, that were indicative of damage to stereocilia and tip links. Finally, the relative susceptibility of hair bundles to overstimulation was addressed by comparing stiffness loss with morphological features in the hair bundles. The loss of stiffness significantly increased as the amount of curvature in the hair bundle contour increased.

Variation in large-conductance, calcium-activated potassium channels from hair cells along the chicken basilar papilla.

The mechanism for electrical tuning in non-mammalian hair cells rests within the widely diverse kinetics of functionally distinct, large-conductance potassium channels (BK), thought to result from alternative splicing of the pore-forming alpha subunit and variable co-expression with an accessory beta subunit. Inside-out patches from hair cells along the chicken basilar papilla revealed 'tonotopic' gradations in calcium sensitivity and deactivation kinetics. The resonant frequency for the hair cell from which the patch was taken was estimated from deactivation rates, and this frequency reasonably matched that predicted from the originating cell's tonotopic location. The rates of deactivation for native BK channels were much faster than rates reported for cloned chicken BK channels including both alpha and beta subunits. This result was surprising since patches were pulled from hair cells in the apical half of the papilla where beta subunits are most highly expressed. Heterogeneity in the properties of native chicken BK channels implies a high degree of molecular variation and hinders our ability to identify those molecular constituents.

Hair bundle profiles along the chick basilar papilla.

Cochlear hair cells play a central role in the transduction of sound into neural output. Anatomical descriptions of these cells, and their protruding hair bundles, are of fundamental interest since hair cell transduction is dependent on hair bundle micromechanics and hair bundle micromechanics depends on hair bundle morphology. In this paper, we describe quantitatively changes in the staircase profile of the hair bundle along the apical portion of the chick's basilar papilla. Images of hair cells from 8 discretely dissected segments of the apical 3rd of the basilar papilla were archived, and the profile contour outlined by the tips of the stereocilia was digitised and curves were fitted by linear and power equations. The hair bundles of tall hair cells exhibited both linear and curvilinear profiles, which were equally distributed along the papilla. All short hair cells in our sample had straight contours. The differences in hair bundle shape among the tall hair cells may lead to differential susceptibility to injury and some variance in the current-displacement transduction curves due to differences in the translation of forces throughout the hair bundle.