Stereociliary Bundles Research Articles

Inner Ear Hair Cell-Like Cells from Human Embryonic Stem Cells

In mammals, the permanence of many forms of hearing loss is the result of the inner ear's inability to replace lost sensory hair cells. Here, we apply a differentiation strategy to guide human embryonic stem cells (hESCs) into cells of the otic lineage using chemically defined attached-substrate conditions. The generation of human otic progenitor cells was dependent on fibroblast growth factor (FGF) signaling, and protracted culture led to the upregulation of markers indicative of differentiated inner ear sensory epithelia. Using a transgenic ESC reporter line based on a murine Atoh1 enhancer, we show that differentiated hair cell-like cells express multiple hair cell markers simultaneously. Hair cell-like cells displayed protrusions reminiscent of stereociliary bundles, but failed to fully mature into cells with typical hair cell cytoarchitecture. We conclude that optimized defined conditions can be used in vitro to attain otic progenitor specification and sensory cell differentiation.

In Vivo Generation of Immature Inner Hair Cells in Neonatal Mouse Cochleae by Ectopic Atoh1 Expression

Regeneration of auditory hair cells (HCs) is a promising approach to restore hearing. Recent studies have demonstrated that induced pluripotent stem cells/embryonic stem cells or supporting cells (SCs) adjacent to HCs can be converted to adopt the HC fate. However, little is known about whether new HCs are characteristic of outer or inner HCs. Here, we showed in vivo conversion of 2 subtypes of SCs, inner border cells (IBs) and inner phalangeal cells (IPhs), to the inner HC (IHC) fate. This was achieved by ectopically activating Atoh1, a transcription factor necessary for HC fate, in IBs/IPhs at birth. Atoh1+ IBs/IPhs first turned on Pou4f3, another HC transcription factor, before expressing 8 HC markers. The conversion rate gradually increased from ∼2.4% at 1 week of age to ∼17.8% in adult. Interestingly, new HCs exhibited IHC characteristics such as straight line–shaped stereociliary bundles, expression of Fgf8 and otoferlin, and presence of larger outward currents than those of outer HCs. However, new HCs lacked the terminal differentiation IHC marker vGlut3, exhibited reduced density of presynaptic Cbtp2 puncta that had little postsynaptic GluR2 specialization, and displayed immature IHC outward currents. Our results demonstrate that the conversion rate of IBs/IPhs in vivo by Atoh1 ectopic expression into the IHC fate was higher and faster and the conversion was more complete than that of the 2 other SC subtypes underneath the outer HCs; however, these new IHCs are arrested before terminal differentiation. Thus, IBs/IPhs are good candidates to regenerate IHCs in vivo.

Force Spectroscopy of Tip Link Proteins: A Study of Inner-Ear Biophysics

Hair cells in the inner ear convert mechanical stimuli, in the vestibular and auditory systems, into electrical signals which can be processed by the brain. Hair cells are highly polarized with a unique elaboration of modified microvilli at their apical surface known as stereo cilia. Sensory stimuli such as sound waves of a particular frequency will result in oscillations of the stereociliary bundle at that frequency. Movements of this bundle couple mechanically to the channel. This force acts to gate the channel in turn resulting in a mechano-electrical transduction. The force is relayed to the channel by filaments known as tip links. Tip links are made of two atypical cadherins, protocadherin-15 and cadherin-23 at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between two anti-parallel pairs of EC domains. Although much has been discerned about the biophysics of this system the lack of adequate technology has prevented the direct measurement of many of these properties. We have developed molecular tools to facilitate the single molecule study of these properties. Self-assembled DNA nanoswitches are functionalized with protocadherin 15 and cadherin 23 fragments using the enzyme sortase. In order to preserve protein function, protein-DNA coupling is performed under physiological conditions. In this two-step process, a small synthetic peptide is first coupled to a DNA oligo. Next, utilizing the enzyme sortase, the protein is coupled to the DNA-peptide chimera under physiological conditions. This strategy frontloads all of the protein-incompatible chemistry so that it is performed on an oligo and a synthetic peptide, which are far more tolerant of non-physiological conditions. Once assembled, the nanoswitches are then used in an optical tweezer system to measure the rupture forces of the tip link under different conditions.

Methionine sulfoxide reductase B3 deficiency causes hearing loss due to stereocilia degeneration and apoptotic cell death in cochlear hair cells

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically reduces methionine-R-sulfoxide to methionine. A recent genetic study showed that the MSRB3 gene is associated with autosomal recessive hearing loss in human deafness DFNB74. However, the precise role of MSRB3 in the auditory system and the pathogenesis of hearing loss have not yet been determined. This work is the first to generate MsrB3 knockout mice to elucidate the possible pathological mechanisms of hearing loss observed in DFNB74 patients. We found that homozygous MsrB3(-/-) mice were profoundly deaf and had largely unaffected vestibular function, whereas heterozygous MsrB3(+/-) mice exhibited normal hearing similar to that of wild-type mice. The MsrB3 protein is expressed in the sensory epithelia of the cochlear and vestibular tissues, beginning at E15.5 and E13.5, respectively. Interestingly, MsrB3 is densely localized at the base of stereocilia on the apical surface of auditory hair cells. MsrB3 deficiency led to progressive degeneration of stereociliary bundles starting at P8, followed by a loss of hair cells, resulting in profound deafness in MsrB3(-/-) mice. The hair cell loss appeared to be mediated by apoptotic cell death, which was measured using TUNEL and caspase 3 immunocytochemistry. Taken together, our data suggest that MsrB3 plays an essential role in maintaining the integrity of hair cells, possibly explaining the pathogenesis of DFNB74 deafness in humans caused by MSRB3 deficiency.

Postnatal Refinement of Auditory Hair Cell Planar Polarity Deficits Occurs in the Absence of Vangl2

The distinctive planar polarity of auditory hair cells is evident in the polarized organization of the stereociliary bundle. Mutations in the core planar cell polarity gene Van Gogh-like 2 (Vangl2) result in hair cells that fail to properly orient their stereociliary bundles along the mediolateral axis of the cochlea. The severity of this phenotype is graded along the length of the cochlea, similar to the hair cell differentiation gradient, suggesting that an active refinement process corrects planar polarity phenotypes in Vangl2 knock-out (KO) mice. Because Vangl2 gene deletions are lethal, Vangl2 conditional knock-outs (CKOs) were generated to test this hypothesis. When crossed with Pax2-Cre, Vangl2 is deleted from the inner ear, yielding planar polarity phenotypes similar to Vangl2 KOs at late embryonic stages except that Vangl2 CKO mice are viable and do not have craniorachischisis like Vangl2 KOs. Quantification of planar polarity deficits through postnatal development demonstrates the activity of a Vangl2-independent refinement process that rescues the planar polarity phenotype within 10 d of birth. In contrast, the Pax2-Cre;Vangl2 CKO has profound changes in the shape and distribution of outer pillar cell and Deiters' cell phalangeal processes that are not corrected during the period of planar polarity refinement. Auditory brainstem response analyses of adult mice show a 10-15 dB shift in auditory threshold, and distortion product otoacoustic emission measurements indicate that this mild hearing deficit is of cochlear origin. Together, these data demonstrate a Vangl2-independent refinement mechanism that actively reorients auditory stereociliary bundles and reveals an unexpected role of Vangl2 during supporting cell morphogenesis.

Conditional Deletion of Atoh1 Reveals Distinct Critical Periods for Survival and Function of Hair Cells in the Organ of Corti

Atonal homolog1 (Atoh1) encodes a basic helix-loop-helix protein that is the first transcription factor to be expressed in differentiating hair cells. Previous work suggests that expression of Atoh1 in prosensory precursors is necessary for the differentiation and survival of hair cells, but it is not clear whether Atoh1 is required exclusively for these processes, or whether it regulates other functions later during hair cell maturation. We used EGFP-tagged Atoh1 knock-in mice to demonstrate for the first time that Atoh1 protein is expressed in hair cell precursors several days before the appearance of differentiated markers, but not in the broad pattern expected of a proneural gene. We conditionally deleted Atoh1 at different points in hair cell development and observe a rapid onset of hair cell defects, suggesting that the Atoh1 protein is unstable in differentiating hair cells and is necessary through an extended phase of their differentiation. Conditional deletion of Atoh1 reveals multiple functions in hair cell survival, maturation of stereociliary bundles, and auditory function. We show the presence of distinct critical periods for Atoh1 in each of these functions, suggesting that Atoh1 may be directly regulating many aspects of hair cell function. Finally, we show that the supporting cell death that accompanies loss of Atoh1 in hair cells is likely caused by the abortive trans-differentiation of supporting cells into hair cells. Together our data suggest that Atoh1 regulates multiple aspects of hair cell development and function.

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.

The Meckel syndrome protein meckelin (TMEM67) is a key regulator of cilia function but is not required for tissue planar polarity

Meckel syndrome (MKS) is a lethal disorder associated with renal cystic disease, encephalocele, ductal plate malformation and polydactyly. MKS is genetically heterogeneous and part of a growing list of syndromes called ciliopathies, disorders resulting from defective cilia. TMEM67 mutation (MKS3) is a major cause of MKS and the related ciliopathy Joubert syndrome, although the complete etiology of the disease is not well understood. To further investigate MKS3, we analyzed phenotypes in the Tmem67 null mouse (bpck) and in zebrafish tmem67 morphants. Phenotypes similar to those in human MKS and other ciliopathy models were observed, with additional eye, skeletal and inner ear abnormalities characterized in the bpck mouse. The observed disorganized stereociliary bundles in the bpck inner ear and the convergent extension defects in zebrafish morphants are similar to those found in planar cell polarity (PCP) mutants, a pathway suggested to be defective in ciliopathies. However, analysis of classical vertebrate PCP readouts in the bpck mouse and ciliary organization analysis in tmem67 morphants did not support a global loss of planar polarity. Canonical Wnt signaling was upregulated in cyst linings and isolated fibroblasts from the bpck mouse, but was unchanged in the retina and cochlea tissue, suggesting that increased Wnt signaling may only be linked to MKS3 phenotypes associated with elevated proliferation. Together, these data suggest that defective cilia loading, but not a global loss of ciliogenesis, basal body docking or PCP signaling leads to dysfunctional cilia in MKS3 tissues.

Electrical tuning and transduction in short hair cells of the chicken auditory papilla

The avian auditory papilla contains two classes of sensory receptor, tall hair cells (THCs) and short hair cells (SHCs), the latter analogous to mammalian outer hair cells with large efferent but sparse afferent innervation. Little is known about the tuning, transduction, or electrical properties of SHCs. To address this problem, we made patch-clamp recordings from hair cells in an isolated chicken basilar papilla preparation at 33°C. We found that SHCs are electrically tuned by a Ca(2+)-activated K(+) current, their resonant frequency varying along the papilla in tandem with that of the THCs, which also exhibit electrical tuning. The tonotopic map for THCs was similar to maps previously described from auditory nerve fiber measurements. SHCs also possess an A-type K(+) current, but electrical tuning was observed only at resting potentials positive to -45 mV, where the A current is inactivated. We predict that the resting potential in vivo is approximately -40 mV, depolarized by a standing inward current through mechanotransducer (MT) channels having a resting open probability of ∼0.26. The resting open probability stems from a low endolymphatic Ca(2+) concentration (0.24 mM) and a high intracellular mobile Ca(2+) buffer concentration, estimated from perforated-patch recordings as equivalent to 0.5 mM BAPTA. The high buffer concentration was confirmed by quantifying parvalbumin-3 and calbindin D-28K with calibrated postembedding immunogold labeling, demonstrating >1 mM calcium-binding sites. Both proteins displayed an apex-to-base gradient matching that in the MT current amplitude, which increased exponentially along the papilla. Stereociliary bundles also labeled heavily with antibodies against the Ca(2+) pump isoform PMCA2a.

Requirement for Dlgh-1 in Planar Cell Polarity and Skeletogenesis during Vertebrate Development

The development of specialized organs is tightly linked to the regulation of cell growth, orientation, migration and adhesion during embryogenesis. In addition, the directed movements of cells and their orientation within the plane of a tissue, termed planar cell polarity (PCP), appear to be crucial for the proper formation of the body plan. In Drosophila embryogenesis, Discs large (dlg) plays a critical role in apical-basal cell polarity, cell adhesion and cell proliferation. Craniofacial defects in mice carrying an insertional mutation in Dlgh-1 suggest that Dlgh-1 is required for vertebrate development. To determine what roles Dlgh-1 plays in vertebrate development, we generated mice carrying a null mutation in Dlgh-1. We found that deletion of Dlgh-1 caused open eyelids, open neural tube, and misorientation of cochlear hair cell stereociliary bundles, indicative of defects in planar cell polarity (PCP). Deletion of Dlgh-1 also caused skeletal defects throughout the embryo. These findings identify novel roles for Dlgh-1 in vertebrates that differ from its well-characterized roles in invertebrates and suggest that the Dlgh-1 null mouse may be a useful animal model to study certain human congenital birth defects.

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca2+ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca2+ permeability, PCa, of the OHC MT channel decreased from cochlear apex to base. This gradient in PCa was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, PCa in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, PCa in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca2+ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher PCa in IHCs and immature apical OHCs. Changes in PCa with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.

Jagged1-mediated Notch signaling regulates mammalian inner ear development independent of lateral inhibition

Conclusion: Jagged1-mediated Notch signaling regulates hair cell (HC) production in a distinct way rather than lateral inhibition mediated by Hes1 and Hes5. Jagged1 may interact with Notch3, probably via candidate downstream mediators Hesr1 and Hesr2, regulating the prosensory formation in the early stage. Objectives: To explore the function of the Jagged1-mediated Notch signaling pathway in mammalian inner ear development and its possible mechanism. Methods: Using conditional gene targeting, a novel Jagged1 conditional knockout (Jag1-cko), Pax8cre/+; Jag1flox/flox, was established. The auditory brainstem response and swim ability test were utilized to identify functional disability. The expression of Jagged1, Notch3, Hes1, Hesr1, and Hesr2 was detected by immunofluorescence and immunohistochemistry. Results: Our Jag1-cko model was established and survived well. It presented hearing impairment and balance disturbance with ‘waltzing' behavior. Cochleae and vestibular apparatus were all found in our Jag1-cko model. Patch deficiency of outer hair cells (OHCs) was found on the apical and middle turns of the auditory epithelium. OHCs were totally missing on the basal turn. The stereociliary bundles were disorientated on the cristae. Unlike Hes1, no expression of Notch3, Hesr1, and Hesr2 was found on embryonic day 13.5 of the Jag1-cko model.

Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.

The development, distribution and density of the plasma membrane calcium ATPase 2 calcium pump in rat cochlear hair cells

Calcium is tightly regulated in cochlear outer hair cells (OHCs). It enters mainly via mechanotransducer (MT) channels and is extruded by the plasma membrane calcium ATPase (PMCA)2 isoform of the PMCA, mutations in which cause hearing loss. To assess how pump expression matches the demands of Ca(2+) homeostasis, the distribution of PMCA2 at different cochlear locations during development was quantified using immunofluorescence and post-embedding immunogold labeling. The PMCA2 isoform was confined to stereociliary bundles, first appearing at the base of the cochlea around post-natal day (P)0 followed by the middle and then the apex by P3, and was unchanged after P8. The developmental appearance matched the maturation of the MT channels in rat OHCs. High-resolution immunogold labeling in adult rats showed that PMCA2 was distributed along the membranes of all three rows of OHC stereocilia at similar densities and at about a quarter of the density in inner hair cell stereocilia. The difference between OHCs and inner hair cells was similar to the ratio of their MT channel resting open probabilities. Gold particle counts revealed no difference in PMCA2 density between low- and high-frequency OHC bundles despite larger MT currents in high-frequency OHCs. The PMCA2 density in OHC stereocilia was determined in low- and high-frequency regions from calibration of immunogold particle counts as 2200/μm(2) from which an extrusion rate of ∼200 ions/s per pump was inferred. The limited ability of PMCA2 to extrude the Ca(2+) load through MT channels may constitute a major cause of OHC vulnerability and high-frequency hearing loss.

PTK7 Regulates Myosin II Activity to Orient Planar Polarity in the Mammalian Auditory Epithelium

Planar cell polarity (PCP) signaling is a key regulator of epithelial morphogenesis, including neural tube closure and the orientation of inner ear sensory hair cells, and is mediated by a conserved noncanonical Wnt pathway. Ptk7 is a novel vertebrate-specific regulator of PCP, yet the mechanisms by which Ptk7 regulates mammalian epithelial PCP remain poorly understood. Here we show that, in the mammalian auditory epithelium, Ptk7 is not required for membrane recruitment of Dishevelled 2; Ptk7 and Frizzled3/Frizzled6 receptors act in parallel and have opposing effects on hair cell PCP. Mosaic analysis identified a requirement of Ptk7 in neighboring supporting cells for hair cell PCP. Ptk7 and the noncanonical Wnt pathway differentially regulate a contractile myosin II network near the apical surface of supporting cells. We provide evidence that this apical myosin II network exerts polarized contractile tension on hair cells to align their PCP, as revealed by asymmetric junctional recruitment of vinculin, a tension-sensitive actin binding protein. In Ptk7 mutants, compromised myosin II activity resulted in loss of planar asymmetry and reduced junctional localization of vinculin. By contrast, vinculin planar asymmetry and stereociliary bundle orientation were restored in Fz3(-/-);Ptk7(-/-) double mutants. These findings suggest that PTK7 acts in conjunction with the noncanonical Wnt pathway to orient epithelial PCP through modulation of myosin II-based contractile tension between supporting cells and hair cells.

Functional Features of Trans-Differentiated Hair Cells Mediated by Atoh1 Reveals a Primordial Mechanism

Evolution has transformed a simple ear with few vestibular maculae into a complex three-dimensional structure consisting of nine distinct endorgans. It is debatable whether the sensory epithelia underwent progressive segregation or emerged from distinct sensory patches. To address these uncertainties we examined the morphological and functional phenotype of trans-differentiated rat hair cells to reveal their primitive or endorgan-specific origins. Additionally, it is uncertain how Atoh1-mediated trans-differentiated hair cells trigger the processes that establish their neural ranking from the vestibulocochlear ganglia. We have demonstrated that the morphology and functional expression of ionic currents in trans-differentiated hair cells resemble those of "ancestral" hair cells, even at the lesser epithelia ridge aspects of the cochlea. The structures of stereociliary bundles of trans-differentiated hair cells were in keeping with cells in the vestibule. Functionally, the transient expression of Na⁺ and I(h) currents initiates and promotes evoked spikes. Additionally, Ca²⁺ current was expressed and underwent developmental changes. These events correlate well with the innervation of ectopic hair cells. New "born" hair cells at the abneural aspects of the cochlea are innervated by spiral ganglion neurons, presumably under the tropic influence of chemoattractants. The disappearance of inward currents coincides well with the attenuation of evoked electrical activity, remarkably recapitulating the development of hair cells. Ectopic hair cells underwent stepwise changes in the magnitude and kinetics of transducer currents. We propose that Atoh1 mediates trans-differentiation of morphological and functional "ancestral" hair cells that are likely to undergo diversification in an endorgan-specific manner.

Nonlinearities in Threshold-Level Detection by Inner Ear Hair Cells

Auditory system is known for its exquisite sensitivity with sub-nanometer detection thresholds. Mechanical deflections due to external sound and ground vibrations are converted by inner ear hair cells into electrical signals. In some species, hair cell's stereociliary bundles exhibit spontaneous oscillations under in vitro conditions, a behavior that has been successfully reproduced by numerical models based on nonlinear dynamics. In this work, we study both numerically and experimentally the dynamics of individual hair bundles from the Bullfrog sacculus, at threshold levels of stimulation to elucidate the mechanisms underlying the sensitivity of detection by the sacculus. We measure the steady state response to small sinusoidal stimuli, as well as the time course of the phase locking transition in spontaneously oscillating bundles.

Self-Tuning of Hair Cells in the Bullfrog Sacculus

Spontaneous oscillations of the stereociliary bundle of a hair cell - the mechanosensory cell in auditory and vestibular systems - is considered to be a signature of an active amplification mechanism. We study whether an internal self-tuning process governs the active motility, by mimicking the effects of loud sound on the spontaneous oscillation. After the application of high-amplitude stimuli, with deflections on the order of micrometers applied to the hair bundle, the active oscillatory motion of the hair bundle was suppressed for hundreds of milliseconds, indicating a change in the dynamic state of the hair cell.

Magnetic Nanoparticles as Mechanical Actuators of Inner Ear Hair Cells

Mechanical sensation by the auditory system is performed by hair cells, named in reference to the stereociliary bundles that protrude from their apical surface. They have been shown to exhibit active motility under in vitro conditions and a highly nonlinear dynamic response. To explore their mechanical properties, we developed a technique that utilizes paramagnetic beads to actuate the stereocilia. Steady-state deflections were imposed and seen to strongly affect the dynamic state of the bundle, inducing a transition from multi-mode to single-mode state, as well as the crossover from the oscillatory to the quiescent state. Numerical simulations capturing the behavior near the critical points will be presented.

Magnetic actuation of hair cells

The bullfrog sacculus contains mechanically sensitive hair cells whose stereociliary bundles oscillate spontaneously when decoupled from the overlying membrane. Steady-state offsets on the resting position of a hair bundle can suppress or modulate this native motility. To probe the dynamics of spontaneous oscillation in the proximity of the critical point, we describe here a method for mechanical actuation that avoids loading the bundles or contributing to the viscous drag. Magnetite beads were attached to the tips of the stereocilia, and a magnetic probe was used to impose deflections. This technique allowed us to observe the transition from multi-mode to single-mode state in freely oscillating bundles, as well as the crossover from the oscillatory to the quiescent state.