Aminoglycosides (AGs) are commonly used antibiotics that cause deafness through the irreversible loss of cochlear sensory hair cells (HCs). How AGs enter the cochlea and then target HCs remains ...unresolved. Here, we performed time-lapse multicellular imaging of cochlea in live adult hearing mice via a chemo-mechanical cochleostomy. The in vivo tracking revealed that systemically administered Texas Red-labeled gentamicin (GTTR) enters the cochlea via the stria vascularis and then HCs selectively. GTTR uptake into HCs was completely abolished in transmembrane channel-like protein 1 (TMC1) knockout mice, indicating mechanotransducer channel-dependent AG uptake. Blockage of megalin, the candidate AG transporter in the stria vascularis, by binding competitor cilastatin prevented GTTR accumulation in HCs. Furthermore, cilastatin treatment markedly reduced AG-induced HC degeneration and hearing loss in vivo. Together, our in vivo real-time tracking of megalin-dependent AG transport across the blood-labyrinth barrier identifies new therapeutic targets for preventing AG-induced ototoxicity.
Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair ...cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss.
Adaptation is a hallmark of hair cell mechanotransduction, extending the sensory hair bundle dynamic range while providing mechanical filtering of incoming sound. In hair cells responsive to low ...frequencies, two distinct adaptation mechanisms exist, a fast component of debatable origin and a slow myosin-based component. It is generally believed that Ca2+ entry through mechano-electric transducer channels is required for both forms of adaptation. This study investigates the calcium dependence of adaptation in the mammalian auditory system. Recordings from rat cochlear hair cells demonstrate that altering Ca2+ entry or internal Ca2+ buffering has little effect on either adaptation kinetics or steady-state adaptation responses. Two additional findings include a voltage-dependent process and an extracellular Ca2+ binding site, both modulating the resting open probability independent of adaptation. These data suggest that slow motor adaptation is negligible in mammalian auditory cells and that the remaining adaptation process is independent of calcium entry.
•Mechanotransduction adaptation does not require calcium•Calcium alters mechanotransduction via an external site•Voltage alters mechanotransducer resting probability of opening•Mechanotransducer adaptation is independent of calcium entry
Adaptation of hair cell mechanotransduction has been thought to be driven by calcium entry via mechanotransducer channels. Here, Peng et al. demonstrate that mammalian cochlear hair cell adaptation does not require calcium entry, arguing against currently held views and prompting consideration of new mechanisms.
Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP
) can directly or indirectly modify ion-channel properties. In ...auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP
localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP
in inner hair-cell stereocilia resulted in the following: (1) the loss of a fast component of mechanoelectric-transduction current adaptation, (2) an increase in the number of channels open at the hair bundle's resting position, (3) a reduction of single-channel conductance, (4) a change in ion selectivity, and (5) a reduction in calcium pore blocking effects. These changes occur without altering hair-bundle compliance or the number of functional stereocilia within a given hair bundle. Although the specific molecular mechanism for PIP
action remains to be uncovered, data support a hypothesis for PIP
directly regulating channel conformation to alter calcium permeation and single-channel conductance.
How forces are relayed to the auditory mechanoelectrical transduction (MET) channel remains unknown. However, researchers have surmised that lipids might be involved. Previous work on bullfrog hair cells showed an effect of phosphoinositol-4,5-bisphosphate (PIP
) depletion on MET current amplitude and adaptation, leading to the postulation of the existence of an underlying myosin-based adaptation mechanism. We find similar results in rat cochlea hair cells but extend these data to include single-channel analysis, hair-bundle mechanics, and channel-permeation properties. These additional data attribute PIP
effects to actions on MET-channel properties and not motor interactions. Further findings support PIP
's role in modulating a fast, myosin-independent, and Ca
-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP
's pivotal role in auditory MET, likely as a direct channel modulator.
In the inner ear, the deflection of hair bundles, the sensory organelles of hair cells, activates mechanically-gated channels (MGCs). Hair bundles monitor orientation of the head, its angular and ...linear acceleration, and detect sound. Force applied to MGCs is shaped by intrinsic hair-bundle properties, by the mechanical load on the bundle, and by the filter imparted by the environment of the hair bundle. Channel gating and adaptation, the ability of the bundle to reset its operating point, contribute to hair-bundle mechanics. Recent data from mammalian hair cells challenge longstanding hypotheses regarding adaptation mechanisms and hair-bundle coherence. Variations between hair bundles from different organs in hair-bundle mechanics, mechanical load, channel gating, and adaptation may allow a hair bundle to selectively respond to specific sensory stimuli.
MGCs, located at the tops of the sensory hair bundles, translate sound as well as the head’s orientation and acceleration into electrical signals.
Mechanical loading of a hair bundle by an accessory structure shapes the response of the bundle to stimulation. Bundles can be freestanding, encased in a cupula, or embedded in a sallet, tectorial membrane, or otolithic membrane.
Hair-bundle compliance and coordination between stereocilia control the macroscopic hair-cell response.
Owing to the sizable gating swing, channel gating affects hair-bundle compliance, creating mechanical nonlinearity.
Adaptation modulates the gating of the MGCs by means of calcium-dependent and independent processes.
Channel gating, adaptation, hair-bundle mechanics, and hair-bundle load determine force transfer to the MGCs, specializing a bundle’s response to distinct types of stimuli.
Hair cells detect vibrations of their stereociliary bundle by activation of mechanically sensitive transducer channels. Although evidence suggests the transducer channels are near the stereociliary ...tops and are opened by force imparted by tip links connecting contiguous stereocilia, the exact channel site remains controversial. We used fast confocal imaging of fluorescence changes reflecting calcium entry during bundle stimulation to localize the channels. Calcium signals were visible in single stereocilia of rat cochlear inner hair cells and were up to tenfold larger and faster in the second and third stereociliary rows than in the tallest first row. The number of functional stereocilia was proportional to transducer current amplitude, indicating that there were about two channels per stereocilium. Comparable results were obtained in outer hair cells. The observations, supported by theoretical simulations, suggest there are no functional mechanically sensitive transducer channels in first row stereocilia and imply the channels are present only at the bottom of the tip links.
In vivo and real-time multicellular imaging enables the decoding of sensory circuits and the tracking of systemic drug uptake. However, in vivo imaging of the auditory periphery remains technically ...challenging owing to the deep location, mechanosensitivity and fluid-filled, bone-encased nature of the cochlear structure. Existing methods that expose the cochlea invariably cause irreversible damage to auditory function, severely limiting the experimental measurements possible in living animals. Here we present an in vivo surgical protocol that permits the imaging of cochlear cells in hearing mice. Our protocol describes a ventro-lateral approach for preserving external and middle ear structures while performing surgery, the correct mouse positioning for imaging cochlear cells with effective sound transmission into the ear, the chemo-mechanical cochleostomy for creating the imaging window in the otic capsule bone that prevents intracochlear fluid leakage by maintaining an intact endosteum, and the release of intracochlear pressure that separates the endosteum from the otic capsule bone while creating an imaging window. The procedure thus preserves hearing thresholds. Individual inner and outer hair cells, supporting cells and nerve fibers can be visualized in vivo while hearing function is preserved. This approach may enable future original investigations, such as the real-time tracking of ototoxic drug transport into the cochleae. The technique may be applied to the monitoring of sound-evoked functional activity in multiple cochlear cells, in combination with optogenetic tools, and may help to improve cochlear implantation in humans. The cochleostomy takes ~1 h and requires experience in surgery.
The auditory system is able to detect movement down to atomic dimensions. This sensitivity comes in part from mechanisms associated with gating of hair cell mechanoelectric transduction (MET) ...channels. MET channels, located at the tops of stereocilia, are poised to detect tension induced by hair bundle deflection. Hair bundle deflection generates a force by pulling on tip-link proteins connecting adjacent stereocilia. The resting open probability (P(open)) of MET channels determines the linearity and sensitivity to mechanical stimulation. Classically, P(open) is regulated by a calcium-sensitive adaptation mechanism in which lowering extracellular calcium or depolarization increases P(open). Recent data demonstrated that the fast component of adaptation is independent of both calcium and voltage, thus requiring an alternative explanation for the sensitivity of P(open) to calcium and voltage. Using rat auditory hair cells, we characterize a mechanism, separate from fast adaptation, whereby divalent ions interacting with the local lipid environment modulate resting P(open). The specificity of this effect for different divalent ions suggests binding sites that are not an EF-hand or calmodulin model. GsMTx4, a lipid-mediated modifier of cationic stretch-activated channels, eliminated the voltage and divalent sensitivity with minimal effects on adaptation. We hypothesize that the dual mechanisms (lipid modulation and adaptation) extend the dynamic range of the system while maintaining adaptation kinetics at their maximal rates.
Mechanosensitive sensory hair cells are the linchpin of our senses of hearing and balance. The inability of the mammalian inner ear to regenerate lost hair cells is the major reason for the ...permanence of hearing loss and certain balance disorders. Here, we present a stepwise guidance protocol starting with mouse embryonic stem and induced pluripotent stem cells, which were directed toward becoming ectoderm capable of responding to otic-inducing growth factors. The resulting otic progenitor cells were subjected to varying differentiation conditions, one of which promoted the organization of the cells into epithelial clusters displaying hair cell-like cells with stereociliary bundles. Bundle-bearing cells in these clusters responded to mechanical stimulation with currents that were reminiscent of immature hair cell transduction currents.
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► ESCs and iPSCs can be guided along the otic lineage ► In vitro-differentiated cells display typical hair cell cytomorphology ► Mechanical responses are reminiscent of hair cell transduction currents
Sensory hair cells are mechanoreceptors required for hearing and balance functions. From embryonic development, hair cells acquire apical stereociliary bundles for mechanosensation, basolateral ion ...channels that shape receptor potential, and synaptic contacts for conveying information centrally. These key maturation steps are sequential and presumed coupled; however, whether hair cells emerging postnatally mature similarly is unknown. Here, we show that in vivo postnatally generated and regenerated hair cells in the utricle, a vestibular organ detecting linear acceleration, acquired some mature somatic features but hair bundles appeared nonfunctional and short. The utricle consists of two hair cell subtypes with distinct morphological, electrophysiological and synaptic features. In both the undamaged and damaged utricle, fate-mapping and electrophysiology experiments showed that Plp1+ supporting cells took on type II hair cell properties based on molecular markers, basolateral conductances and synaptic properties yet stereociliary bundles were absent, or small and nonfunctional. By contrast, Lgr5+ supporting cells regenerated hair cells with type I and II properties, representing a distinct hair cell precursor subtype. Lastly, direct physiological measurements showed that utricular function abolished by damage was partially regained during regeneration. Together, our data reveal a previously unrecognized aberrant maturation program for hair cells generated and regenerated postnatally and may have broad implications for inner ear regenerative therapies.
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