The Cousa objective: a long-working distance air objective for multiphoton imaging in vivo.

Multiphoton microscopy can resolve fluorescent structures and dynamics deep in scattering tissue and has transformed neural imaging, but applying this technique in vivo can be limited by the mechanical and optical constraints of conventional objectives. Short working distance objectives can collide with compact surgical windows or other instrumentation and preclude imaging. Here we present an ultra-long working distance (20 mm) air objective called the Cousa objective. It is optimized for performance across multiphoton imaging wavelengths, offers a more than 4 mm2 field of view with submicrometer lateral resolution and is compatible with commonly used multiphoton imaging systems. A novel mechanical design, wider than typical microscope objectives, enabled this combination of specifications. We share the full optical prescription, and report performance including in vivo two-photon and three-photon imaging in an array of species and preparations, including nonhuman primates. The Cousa objective can enable a range of experiments in neuroscience and beyond.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells.

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.

In vivo real-time imaging reveals megalin as the aminoglycoside gentamicin transporter into cochlea whose inhibition is otoprotective.

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.

Coupling between the Stereocilia of Rat Sensory Inner-Hair-Cell Hair Bundles Is Weak, Shaping Their Sensitivity to Stimulation.

The hair bundle is the universal mechanosensory organelle of auditory, vestibular, and lateral-line systems. A bundle comprises mechanically coupled stereocilia, whose displacements in response to stimulation activate a receptor current. The similarity of stereociliary displacements within a bundle regulates fundamental properties of the receptor current like its speed, magnitude, and sensitivity. However, the dynamics of individual stereocilia from the mammalian cochlea in response to a known bundle stimulus has not been quantified. We developed a novel high-speed system, which dynamically stimulates and tracks individual inner-hair-cell stereocilia from male and female rats. Stimulating two to three of the tallest stereocilia within a bundle (nonuniform stimulation) caused dissimilar stereociliary displacements. Stereocilia farther from the stimulator moved less, but with little delay, implying that there is little slack in the system. Along the axis of mechanical sensitivity, stereocilium displacements peaked and reversed direction in response to a step stimulus. A viscoelastic model explained the observed displacement dynamics, which implies that coupling between the tallest stereocilia is effectively viscoelastic. Coupling elements between the tallest inner-hair-cell stereocilia were two to three times stronger than elements anchoring stereocilia to the surface of the cell but were 100-10,000 times weaker than those of a well-studied noncochlear hair bundle. Coupling was too weak to ensure that stereocilia move similarly in response to nonuniform stimulation at auditory frequencies. Our results imply that more uniform stimulation across the tallest stereocilia of an inner-hair-cell bundle in vivo is required to ensure stereociliary displacement similarity, increasing the speed, sensitivity, and magnitude of the receptor current.SIGNIFICANCE STATEMENT Generation of the receptor current of the hair cell is the first step in electrically encoding auditory information in the hearing organs of all vertebrates. The receptor current is shaped by mechanical coupling between stereocilia in the hair bundle of each hair cell. Here, we provide foundational information on the mechanical coupling between stereocilia of cochlear inner-hair cells. In contrast to other types of hair cell, coupling between inner-hair-cell stereocilia is weak, causing slower, smaller, and less sensitive receptor currents in response to stimulation of few, rather than many, stereocilia. Our results imply that inner-hair cells need many stereocilia to be stimulated in vivo to ensure fast, large, and sensitive receptor currents.

Identifying targets to prevent aminoglycoside ototoxicity.

Aminoglycosides are potent antibiotics that are commonly prescribed worldwide. Their use carries significant risks of ototoxicity by directly causing inner ear hair cell degeneration. Despite their ototoxic side effects, there are currently no approved antidotes. Here we review recent advances in our understanding of aminoglycoside ototoxicity, mechanisms of drug transport, and promising sites for intervention to prevent ototoxicity.

Dissociating antibacterial from ototoxic effects of gentamicin C-subtypes.

Gentamicin is a potent broad-spectrum aminoglycoside antibiotic whose use is hampered by ototoxic side-effects. Hospital gentamicin is a mixture of five gentamicin C-subtypes and several impurities of various ranges of nonexact concentrations. We developed a purification strategy enabling assaying of individual C-subtypes and impurities for ototoxicity and antimicrobial activity. We found that C-subtypes displayed broad and potent in vitro antimicrobial activities comparable to the hospital gentamicin mixture. In contrast, they showed different degrees of ototoxicity in cochlear explants, with gentamicin C2b being the least and gentamicin C2 the most ototoxic. Structure-activity relationships identified sites in the C4'-C6' region on ring I that reduced ototoxicity while preserving antimicrobial activity, thus identifying targets for future drug design and mechanisms for hair cell toxicity. Structure-activity relationship data suggested and electrophysiological data showed that the C-subtypes both bind and permeate the hair cell mechanotransducer channel, with the stronger the binding the less ototoxic the compound. Finally, both individual and reformulated mixtures of C-subtypes demonstrated decreased ototoxicity while maintaining antimicrobial activity, thereby serving as a proof-of-concept of drug reformulation to minimizing ototoxicity of gentamicin in patients.

Loxhd1 Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells.

Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase α-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the mechanotransduction phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphologic defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multirepeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.

Uncoordinated maturation of developing and regenerating postnatal mammalian vestibular hair cells.

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.

Functional subgroups of cochlear inner hair cell ribbon synapses differently modulate their EPSC properties in response to stimulation.

Spiral ganglion neurons (SGNs) form single synapses on inner hair cells (IHCs), transforming sound-induced IHC receptor potentials into trains of action potentials. SGN neurons are classified by spontaneous firing rates as well as their threshold response to sound intensity levels. We investigated the hypothesis that synaptic specializations underlie mouse SGN response properties and vary with pillar versus modiloar synapse location around the hair cell. Depolarizing hair cells with 40 mM K+ increased the rate of postsynaptic responses. Pillar synapses matured later than modiolar synapses. Excitatory postsynaptic current (EPSC) amplitude, area, and number of underlying events per EPSC were similar between synapse locations at steady state. However, modiolar synapses produced larger monophasic EPSCs when EPSC rates were low and EPSCs became more multiphasic and smaller in amplitude when rates were higher, while pillar synapses produced more monophasic and larger EPSCs when the release rates were higher. We propose that pillar and modiolar synapses have different operating points. Our data provide insight into underlying mechanisms regulating EPSC generation.NEW & NOTEWORTHY Data presented here provide the first direct functional evidence of late synaptic maturation of the hair cell- spiral ganglion neuron synapse, where pillar synapses mature after postnatal day 20. Data identify a presynaptic difference in release during stimulation. This difference may in part drive afferent firing properties.

Hair Bundle Stimulation Mode Modifies Manifestations of Mechanotransduction Adaptation.

Sound detection in auditory sensory hair cells depends on the deflection of the stereocilia hair bundle which opens mechano-electric transduction (MET) channels. Adaptation is hypothesized to be a critical property of MET that contributes to the auditory system's wide dynamic range and sharp frequency selectivity. Our recent work using a stiff probe to displace hair bundles showed that the fastest adaptation mechanism (fast adaptation) does not require calcium entry. Using fluid-jet stimuli, others obtained data showing only a calcium-dependent fast adaptation response. Because cochlear stereocilia do not move coherently and the hair cell response depends critically on the magnitude and time course of the hair bundle deflection, we developed a high-speed imaging technique to quantify this deflection in rat cochlear hair cells. The fluid jet delivers a force stimulus, and force steps lead to a complex time course of hair bundle displacement (mechanical creep), which affects the hair cell's macroscopic MET current response by masking the time course of the fast adaptation response. Modifying the fluid-jet stimulus to generate a hair bundle displacement step produced rapidly adapting currents that did not depend on membrane potential, confirming that fast adaptation does not depend on calcium entry. MET current responses differ with stimulus modality and will shape receptor potentials of different hair cell types based on their in vivo stimulus mode. These transformations will directly affect how stimuli are encoded.SIGNIFICANCE STATEMENT Mechanotransduction by sensory hair cells represents a key first step for the sound sensing ability in vertebrates. The sharp frequency tuning and wide dynamic range of sound sensation are hypothesized to require a mechanotransduction adaptation mechanism. Recent work indicated that the apparent calcium dependence of the fastest adaptation differs with the method of cochlear hair cell stimulation. Here, we reconcile existing data and show that calcium entry does not drive the fastest adaptation process, independent of the stimulation method. With force stimulation of the hair bundle, adaptation manifests differently than with displacement stimulation, indicating that the stimulation mode of the hair bundle will affect the hair cell receptor current and stimulus coding.

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature.

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

Development and localization of reverse-polarity mechanotransducer channels in cochlear hair cells.

Cochlear hair cells normally detect positive deflections of their hair bundles, rotating toward their tallest edge, which opens mechanotransducer (MT) channels by increased tension in interciliary tip links. After tip-link destruction, the normal polarity of MT current is replaced by a mechanically sensitive current evoked by negative bundle deflections. The "reverse-polarity" current was investigated in cochlear hair cells after tip-link destruction with BAPTA, in transmembrane channel-like protein isoforms 1/2 (Tmc1:Tmc2) double mutants, and during perinatal development. This current is a natural adjunct of embryonic development, present in all wild-type hair cells but declining after birth with emergence of the normal-polarity current. Evidence indicated the reverse-polarity current seen developmentally was a manifestation of the same ion channel as that evident under abnormal conditions in Tmc mutants or after tip-link destruction. In all cases, sinusoidal fluid-jet stimuli from different orientations suggested the underlying channels were opened not directly by deflections of the hair bundle but by deformation of the apical plasma membrane. Cell-attached patch recording on the hair-cell apical membrane revealed, after BAPTA treatment or during perinatal development, 90-pS stretch-activated cation channels that could be blocked by Ca(2+) and by FM1-43. High-speed Ca(2+) imaging, using swept-field confocal microscopy, showed the Ca(2+) influx through the reverse-polarity channels was not localized to the hair bundle, but distributed across the apical plasma membrane. These reverse-polarity channels, which we propose to be renamed "unconventional" mechanically sensitive channels, have some properties similar to the normal MT channels, but the relationship between the two types is still not well defined.

Phosphoinositol-4,5-Bisphosphate Regulates Auditory Hair-Cell Mechanotransduction-Channel Pore Properties and Fast Adaptation.

Membrane proteins, such as ion channels, interact dynamically with their lipid environment. Phosphoinositol-4,5-bisphosphate (PIP2) can directly or indirectly modify ion-channel properties. In auditory sensory hair cells of rats (Sprague Dawley) of either sex, PIP2 localizes within stereocilia, near stereocilia tips. Modulating the amount of free PIP2 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 PIP2 action remains to be uncovered, data support a hypothesis for PIP2 directly regulating channel conformation to alter calcium permeation and single-channel conductance.SIGNIFICANCE STATEMENT 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 (PIP2) 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 PIP2 effects to actions on MET-channel properties and not motor interactions. Further findings support PIP2's role in modulating a fast, myosin-independent, and Ca2+-independent adaptation process, validating fast adaptation's biological origin. Together this shows PIP2's pivotal role in auditory MET, likely as a direct channel modulator.

Adaptation Independent Modulation of Auditory Hair Cell Mechanotransduction Channel Open Probability Implicates a Role for the Lipid Bilayer.

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.

Calcium-induced calcium release supports recruitment of synaptic vesicles in auditory hair cells.

Hair cells from auditory and vestibular systems transmit continuous sound and balance information to the central nervous system through the release of synaptic vesicles at ribbon synapses. The high activity experienced by hair cells requires a unique mechanism to sustain recruitment and replenishment of synaptic vesicles for continuous release. Using pre- and postsynaptic electrophysiological recordings, we explored the potential contribution of calcium-induced calcium release (CICR) in modulating the recruitment of vesicles to auditory hair cell ribbon synapses. Pharmacological manipulation of CICR with agents targeting endoplasmic reticulum calcium stores reduced both spontaneous postsynaptic multiunit activity and the frequency of excitatory postsynaptic currents (EPSCs). Pharmacological treatments had no effect on hair cell resting potential or activation curves for calcium and potassium channels. However, these drugs exerted a reduction in vesicle release measured by dual-sine capacitance methods. In addition, calcium substitution by barium reduced release efficacy by delaying release onset and diminishing vesicle recruitment. Together these results demonstrate a role for calcium stores in hair cell ribbon synaptic transmission and suggest a novel contribution of CICR in hair cell vesicle recruitment. We hypothesize that calcium entry via calcium channels is tightly regulated to control timing of vesicle fusion at the synapse, whereas CICR is used to maintain a tonic calcium signal to modulate vesicle trafficking.

Thyroid hormone is required for pruning, functioning and long-term maintenance of afferent inner hair cell synapses.

Functional maturation of afferent synaptic connections to inner hair cells (IHCs) involves pruning of excess synapses formed during development, as well as the strengthening and survival of the retained synapses. These events take place during the thyroid hormone (TH)-critical period of cochlear development, which is in the perinatal period for mice and in the third trimester for humans. Here, we used the hypothyroid Snell dwarf mouse (Pit1(dw)) as a model to study the role of TH in afferent type I synaptic refinement and functional maturation. We observed defects in afferent synaptic pruning and delays in calcium channel clustering in the IHCs of Pit1(dw) mice. Nevertheless, calcium currents and capacitance reached near normal levels in Pit1(dw) IHCs by the age of onset of hearing, despite the excess number of retained synapses. We restored normal synaptic pruning in Pit1(dw) IHCs by supplementing with TH from postnatal day (P)3 to P8, establishing this window as being critical for TH action on this process. Afferent terminals of older Pit1(dw) IHCs showed evidence of excitotoxic damage accompanied by a concomitant reduction in the levels of the glial glutamate transporter, GLAST. Our results indicate that a lack of TH during a critical period of inner ear development causes defects in pruning and long-term homeostatic maintenance of afferent synapses.

Underestimated sensitivity of mammalian cochlear hair cells due to splay between stereociliary columns.

Current-displacement (I-X) and the force-displacement (F-X) relationships characterize hair-cell mechano-transduction in the inner ear. A common technique for measuring these relationships is to deliver mechanical stimulations to individual hair bundles with microprobes and measure whole cell transduction currents through patch pipette electrodes at the basolateral membrane. The sensitivity of hair-cell mechano-transduction is determined by two fundamental biophysical properties of the mechano-transduction channel, the stiffness of the putative gating spring and the gating swing, which are derived from the I-X and F-X relationships. Although the hair-cell stereocilia in vivo deflect <100 nm even at high sound pressure levels, often it takes >500 nm of stereocilia displacement to saturate hair-cell mechano-transduction in experiments with individual hair cells in vitro. Despite such discrepancy between in vivo and in vitro data, key biophysical properties of hair-cell mechano-transduction to define the transduction sensitivity have been estimated from in vitro experiments. Using three-dimensional finite-element methods, we modeled an inner hair-cell and an outer hair-cell stereocilia bundle and simulated the effect of probe stimulation. Unlike the natural situation where the tectorial membrane stimulates hair-cell stereocilia evenly, probes deflect stereocilia unevenly. Because of uneven stimulation, 1) the operating range (the 10-90% width of the I-X relationship) increases by a factor of 2-8 depending on probe shapes, 2) the I-X relationship changes from a symmetric to an asymmetric function, and 3) the bundle stiffness is underestimated. Our results indicate that the generally accepted assumption of parallel stimulation leads to an overestimation of the gating swing and underestimation of the gating spring stiffness by an order of magnitude.

The how and why of identifying the hair cell mechano-electrical transduction channel.

Identification of the auditory hair cell mechano-electrical transduction (hcMET) channel has been a major focus in the hearing research field since the 1980s when direct mechanical gating of a transduction channel was proposed (Corey and Hudspeth J Neurosci 3:962-976, 1983). To this day, the molecular identity of this channel remains controversial. However, many of the hcMET channel's properties have been characterized, including pore properties, calcium-dependent ion permeability, rectification, and single channel conductance. At this point, elucidating the molecular identity of the hcMET channel will provide new tools for understanding the mechanotransduction process. This review discusses the significance of identifying the hcMET channel, the difficulties associated with that task, as well as the establishment of clear criteria for this identification. Finally, we discuss potential candidate channels in light of these criteria.

Activity-dependent regulation of prestin expression in mouse outer hair cells.

Prestin is a membrane protein necessary for outer hair cell (OHC) electromotility and normal hearing. Its regulatory mechanisms are unknown. Several mouse models of hearing loss demonstrate increased prestin, inspiring us to investigate how hearing loss might feedback onto OHCs. To test whether centrally mediated feedback regulates prestin, we developed a novel model of inner hair cell loss. Injection of diphtheria toxin (DT) into adult CBA mice produced significant loss of inner hair cells without affecting OHCs. Thus, DT-injected mice were deaf because they had no afferent auditory input despite OHCs continuing to receive normal auditory mechanical stimulation and having normal function. Patch-clamp experiments demonstrated no change in OHC prestin, indicating that loss of information transfer centrally did not alter prestin expression. To test whether local mechanical feedback regulates prestin, we used Tecta(C1509G) mice, where the tectorial membrane is malformed and only some OHCs are stimulated. OHCs connected to the tectorial membrane had normal prestin levels, whereas OHCs not connected to the tectorial membrane had elevated prestin levels, supporting an activity-dependent model. To test whether the endocochlear potential was necessary for prestin regulation, we studied Tecta(C1509G) mice at different developmental ages. OHCs not connected to the tectorial membrane had lower than normal prestin levels before the onset of the endocochlear potential and higher than normal prestin levels after the onset of the endocochlear potential. Taken together, these data indicate that OHC prestin levels are regulated through local feedback that requires mechanoelectrical transduction currents. This adaptation may serve to compensate for variations in the local mechanical environment.