Vestibular Hair Cells Research Articles

The Septate Junction Protein Caspr Is Required for Structural Support and Retention of KCNQ4 at Calyceal Synapses of Vestibular Hair Cells

The Septate Junction Protein Caspr Is Required for Structural Support and Retention of KCNQ4 at Calyceal Synapses of Vestibular Hair Cells

Potassium currents induced by hydrostatic pressure modulate membrane potential and transmitter release in vestibular type II hair cells

Vestibular type II hair cells respond to increases in the hydrostatic pressure with pressure-dependent K(+) currents. We examined whether such currents may modulate transmitter release (assessed as membrane capacitance increments) by altering membrane potentials and voltage-gated Ca(2+) currents. Capacitance increments were dependent on voltage-gated Ca(2+) influx. Stimulating currents (0.7 nA) in current clamp induced depolarisations that were more negative by 8.7 +/- 2.1 mV when the bath height was elevated from 0.2 to 0.5 cm. In voltage clamp, protocols were used that simulated the time course of the membrane potential in current clamp at either low (control) or high hydrostatic pressure (high bath). The low bath protocol induced significantly larger Ca(2+) currents and increases in capacitance than the high bath protocol. We conclude that pressure-dependent K(+) currents may alter the voltage response of vestibular hair cells to an extent critical for Ca(2+) currents and transmitter release. This mechanism may contribute to vestibular dysfunction in Meniere's disease.

Gentamicin vestibulotoxicity impairs human electrically evoked vestibulo-ocular reflex

Electrical vestibular stimulation is believed to directly activate the vestibular afferents to mediate an electrically evoked vestibulo-ocular reflex (eVOR). Gentamicin, an aminoglycoside antibiotic, induces vestibulotoxicity by hair cell damage and death. To determine if human eVOR is impaired by hair cell damage and death in systemic gentamicin vestibulotoxicity (GV). Three-dimensional binocular eye movements evoked by bilateral, bipolar, 100 msec direct current-step at intensities of 0.9, 2.5, 5.0, 7.5, and 10.0 mA were recorded with dual-search coils in 12 GV patients, and the results were compared to 13 healthy subjects. Normal eVOR was predominantly torsional, comprising phasic eVOR initiation and cessation acceleration pulses at 9 msec latency after current onset and offset, with a tonic eVOR velocity-step during the 100 msec intervening period of maintained current. Normal phasic eVOR increased, while tonic eVOR scaled linearly, with current intensity. GV impaired phasic eVOR more severely than tonic eVOR, and prolonged the latency to 12-13 msec. In patients without mechanical response to vestibular tests, phasic eVOR was reduced to one-fifth of normal amplitude, doubled in duration, had reduced ability to vary with current intensity, and threshold was increased. Tonic eVOR was reduced to one-third of normal, but still scaled linearly with current intensity. Patients, who retained partial mechanical responses to vestibular tests, had phasic eVOR impairment without tonic eVOR abnormality. Impairment of evoked vestibulo-ocular reflex (eVOR) in gentamicin vestibulotoxicity (GV) suggests that vestibular hair cells, activated by electrical stimulation, mediate the eVOR. Abnormalities of the eVOR, especially the phasic component, might be a marker of vestibular injury in GV.

The muscarinic inhibition of the potassium M-current modulates the action-potential discharge in the vestibular primary-afferent neurons of the rat

There is consensus that muscarinic and nicotinic receptors expressed in vestibular hair cells and afferent neurons are involved in the efferent modulation of the electrical activity of the afferent neurons. However the underlying mechanisms of postsynaptic control in neurons are not well understood. In our work we show that the activation of muscarinic receptors in the vestibular neurons modulates the potassium M-current modifying the activity of afferent neurons.Whole-cell patch-clamp recordings were made on vestibular-afferent neurons isolated from Wistar rats (postnatal days 7–10) and held in primary culture (18–24 h). The M-current was studied during its deactivation after depolarizing voltage-clamp pulses. In 68% of the cells studied, those of larger capacitance, the M-current antagonists linopirdine and XE-991 reduced the amplitude of the M-current by 54%±7% and 50%±3%. The muscarinic-receptor agonist oxotremorine-M also significantly reduced the M-current by 58%±12% in the cells. The action of oxotremorine-M was blocked by atropine, thus indicating its cholinergic nature. The erg-channel blocker E-4031 did not significantly modify the M-current amplitude. In current-clamp experiments, linopirdine, XE-991, and oxotremorine-M modified the discharge response to current pulses from single spike to multiple spiking, reducing the adaptation of the electrical discharge. Our results indicate that large soma-size cultured vestibular-afferent neurons (most probably calyx-bearing neurons) express the M-current and that the modulation of this current by activation of muscarinic-receptor reduces its spike-frequency adaptation.

Endocochlear potential depends on Cl− channels: mechanism underlying deafness in Bartter syndrome IV

Human Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a beta-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in mice now reveals that the positive potential, but not the high potassium concentration, of the scala media depends on the presence of these channels in the epithelium of the stria vascularis. The reduced driving force for K(+)-entry through mechanosensitive channels into sensory hair cells entails a profound congenital hearing loss and subtle vestibular symptoms. Although retaining all cell types and intact tight junctions, the thickness of the stria is reduced early on. Cochlear outer hair cells degenerate over several months. A collapse of endolymphatic space was seen when mice had additionally renal salt and fluid loss due to partial barttin deletion in the kidney. Bsnd(-/-) mice thus demonstrate a novel function of Cl(-) channels in generating the endocochlear potential and reveal the mechanism leading to deafness in human Bartter syndrome IV.

Molecular Identity and Functional Properties of a Novel T-Type Ca2+ Channel Cloned From the Sensory Epithelia of the Mouse Inner Ear

The molecular identity of non-Cav1.3 channels in auditory and vestibular hair cells has remained obscure, yet the evidence in support of their roles to promote diverse Ca2+-dependent functions is indisputable. Recently, a transient Cav3.1 current that serves as a functional signature for the development and regeneration of hair cells has been identified in the chicken basilar papilla. The Cav3.1 current promotes spontaneous activity of the developing hair cell, which may be essential for synapse formation. Here, we have isolated and sequenced the full-length complementary DNA of a distinct isoform of Cav3.1 in the mouse inner ear. The channel is derived from alternative splicing of exon14, exon25A, exon34, and exon35. Functional expression of the channel in Xenopus oocytes yielded Ca2+ currents, which have a permeation phenotype consistent with T-type channels. However, unlike most multiion channels, the T-type channel does not exhibit the anomalous mole fraction effect, possibly reflecting comparable permeation properties of divalent cations. The Cav3.1 channel was expressed in sensory and nonsensory epithelia of the inner ear. Moreover, there are profound changes in the expression levels during development. The differential expression of the channel during development and the pharmacology of the inner ear Cav3.1 channel may have contributed to the difficulties associated with identification of the non-Cav1.3 currents.

State-of-the-art mechanisms of intracochlear drug delivery

Treatment of auditory and vestibular dysfunction has become increasingly dependent on inner ear drug delivery. Recent advances in molecular therapy and nanotechnology have pushed development of alternate delivery methodologies involving both transtympanic and direct intracochlear infusions. This review examines recent developments in the field relevant to both clinical and animal research environments. Transtympanic delivery of gentamicin and corticosteroids for the treatment of Meniere's disease and sudden sensorineural hearing loss continues to be clinically relevant, with understanding of pharmacokinetics becoming more closely studied. Stabilizing matrices placed on the round window membrane for sustained passive delivery of compounds offer more controlled dosing profiles than transtympanic injections. Nanoparticles are capable of traversing the round window membrane and cochlear membranous partitions, and may become useful gene delivery platforms. Cochlear and vestibular hair cell regeneration has been demonstrated by vector delivery to the inner ear, offering promise for future advanced therapies. Optimal methods of inner ear drug delivery will depend on toxicity, therapeutic dose range, and characteristics of the agent to be delivered. Advanced therapy development will likely require direct intracochlear delivery with detailed understanding of associated pharmacokinetics.

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.

Comprehensive Wnt‐related gene expression during cochlear duct development in chicken

The avian cochlear duct houses both a vestibular and auditory sensory organ (the lagena macula and basilar papilla, respectively), which each have a distinct structure and function. Comparative mRNA in situ hybridization mapping conducted over the time course of chicken cochlear duct development reveals that Wnt-related gene expression is concomitant with various developmental processes such as regionalization, convergent extension of the cochlear duct, cell fate specification, synaptogenesis, and the establishment of planar cell polarity. Wnts mostly originate from nonsensory tissue domains, whereas the sensory primordia preferentially transcribe Frizzled receptors, suggesting that paracrine Wnt signaling predominates in the cochlear duct. Superimposed over this is the strong expression of two secreted Frizzled-related Wnt inhibitors that tend to show complementary expression patterns. Frzb (SFRP3) is confined to the nonsensory cochlear duct and the lagena macula, whereas SFRP2 is maintained in the basilar papilla along with Fzd10 and Wnt7b. Flanking the basilar papilla are Wnt7a, Wnt9a, Wnt11, and SFRP2 on the neural side and Wnt5a, Wnt5b, and Wnt7a on the abneural side. The lateral nonsensory cochlear duct continuously expresses Frzb and temporarily expresses Wnt6 and SFRP1. Characteristic for the entire lagena is the expression of Frzb; in the lagena macula are Fzd1, Fzd7, and Wnt7b, and in the nonsensory tissues are Wnt4 and Wnt5a. Auditory hair cells preferentially express Fzd2 and Fzd9, whereas the main receptors expressed in vestibular hair cells are Fzd1 and Fzd7, in addition to Fzd2 and Fzd9.

Resting Discharge Patterns of Macular Primary Afferents in Otoconia-Deficient Mice

Vestibular primary afferents in the normal mammal are spontaneously active. The consensus hypothesis states that such discharge patterns are independent of stimulation and depend instead on excitation by vestibular hair cells due to background release of synaptic neurotransmitter. In the case of otoconial sensory receptors, it is difficult to test the independence of resting discharge from natural tonic stimulation by gravity. We examined this question by studying discharge patterns of single vestibular primary afferent neurons in the absence of gravity stimulation using two mutant strains of mice that lack otoconia (OTO-; head tilt, het-Nox3, and tilted, tlt-Otop1). Our findings demonstrated that macular primary afferent neurons exhibit robust resting discharge activity in OTO- mice. Spike interval coefficient of variation (CV = SD/mean spike interval) values reflected both regular and irregular discharge patterns in OTO- mice, and the range of values for rate-normalized CV was similar to mice and other mammals with intact otoconia although there were proportionately fewer irregular fibers. Mean discharge rates were slightly higher in otoconia-deficient strains even after accounting for proportionately fewer irregular fibers [OTO- = 75.4 +/- 31.1(113) vs OTO+ = 68.1 +/- 28.5(143) in sp/s]. These results confirm the hypothesis that resting activity in macular primary afferents occurs in the absence of ambient stimulation. The robust discharge rates are interesting in that they may reflect the presence of a functionally 'up-regulated' tonic excitatory process in the absence of natural sensory stimulation.

Role of proinflammatory cytokines in cisplatin‐induced vestibular hair cell damage

Cisplatin causes the impairment of inner ear functions, including hearing and balance, through the involvement of a number of mechanisms. However, no laboratory studies have been performed on involvement of inflammation-related events in cisplatin-mediated vestibular dysfunction. We evaluated the secretion of proinflammatory cytokines and nuclear factor-kappaB (NF-kappaB) activation in cisplatin-treated UB/UE-1 utricular epithelial cells. We also employed immunohistochemistry to detect proinflammatory cytokines and NF-kappaB expression in cisplatin-injected mice. Productions of proinflammatory cytokines significantly caused the death of UB/UE1 cells by cisplatin. Pharmacologic inhibition of mitogen-activated protein (MAP) kinase/ERK kinase-1 (MEK1) or extracellular signal-regulated kinase (ERK) significantly attenuated the death of UB/UE1 cells caused by cisplatin and proinflammatory cytokines. Immunohistochemical studies revealed an increase in the expression of proinflammatory cytokines and NF-kappaB in both the cristae ampullae and utricle of cisplatin-injected mice. These results suggest that proinflammatory cytokines may play an important role in the pathogenesis of cisplatin-mediated vestibulo-toxicity.

Osteopontin is not Critical for Otoconia Formation or Balance Function

Unlike the structural and mechanical role of bone crystals, the inertial mass of otoconia crystals provides a shearing force to stimulate the mechanoreceptors of the utricle and saccule (the gravity receptor organ) under the stimuli of linear motion. It is not clear whether otoconia, composed primarily of CaCO3 and glycoproteins, go through similar calcification processes as bone. We have recently shown that otoconin-90 (Oc90) regulates the growth of otoconia crystals as osteopontin does bone crystals. Here, we analyzed the role of this non-collagenous bone matrix protein, osteopontin, in otoconia formation and balance function utilizing its knockout mice, whose inner ear phenotype has not been examined. Despite the presence of the protein in wild-type otoconia and vestibular hair cells, morphological, ultrastructural, and protein and calcium composition analyses of osteopontin null otoconia show that the protein is not needed for crystal formation, and no evidence of compensatory protein deposition is found. Employment of a wide spectrum of balance behavioral tests demonstrates that the protein is not critical for balance function either, which is confirmed by the normal function of the gravity receptor organ directly measured with linear vestibular-evoked potentials (VsEPs). When compared with findings on other otoconins, the data manifest a hierarchy of importance of proteins in crystallization and indicate mechanistic similarities and differences between bone and otoconia calcification.

A locus on distal chromosome 10 ( ahl4) affecting age-related hearing loss in A/J mice

The ahl locus, shown to be a strain-specific Cdh23 dimorphism, contributes to age-related hearing loss in many inbred mouse strains. A/J mice begin to lose hearing by 4 weeks of age, much earlier than C57BL/6J (B6) mice, although both strains have the same Cdh23 ahl variant. Here, we use recombinant inbred strains, chromosome substitution strains, and a linkage backcross to map a locus on distal Chromosome 10, designated ahl4, that contributes to the early-onset hearing loss of A/J mice. Cochleae of 9-week-old A/J mice exhibit inner and outer hair cell loss from the basal turn through the apical turn, with outer hair cell loss at the base being severest. To quantify the progression of hair cell loss, cytocochleograms were evaluated from 0 to 20 weeks of age. A/J mice showed evidence of hair cell loss in the base of the cochlea as early as 14 days of age and the magnitude and extent of loss increased rapidly during the following 2–5 months. Hair cell loss occurred earlier and was much more severe and widespread in A/J mice than in B6 mice during the first 5 months of age. Spiral ganglion neurons, cells of the stria vascularis, and vestibular hair cell densities, however, appeared normal in 20-week-old A/J mice.

Otoconin-90 deletion leads to imbalance but normal hearing: A comparison with other otoconia mutants

Our sense of gravitation and linear acceleration is mediated by stimulation of vestibular hair cells through displacement of otoconia in the utricle and saccule (the gravity receptor organ). We recently showed that otoconin-90 (Oc90) deletion led to formation of giant otoconia. In the present study, we determined the extent to which the giant otoconia affected balance and gravity receptor sensory input and compared the findings with other otoconia mutants. We employed a wide spectrum of balance behavioral tests, including reaching and air-righting reflexes, gait, swimming, beam-crossing, rotorod latencies, and a direct measure of gravity receptor input, vestibular evoked potentials (VsEPs). All tests on homozygous adult mutants consistently ranked the order of imbalance as (from worst to best) Nox3 het<otopetrin 1 tlt<Oc90 null<Oc90 wild type and C57Bl/6 mice using systematic statistical comparisons of the frequency of occurrence or the severity of abnormal functions. This order coincides with the degree of otoconia deficiencies and is consistent with VsEP measures. Notably, all mice (except Nox3 het) showed remarkable learned adaptation to peripheral vestibular deficits by staying on the rotating rod significantly longer in each successive trial, and the rate and extent of such learned improvements ranked the same order as their initial balance ability. Despite the vestibular morbidity, Oc90 null mice had normal hearing, as measured by auditory brainstem responses (ABRs) and distortion products of otoacoustic emissions (DPOAEs). The study demonstrates that the remnant otoconia mass in Oc90 nulls does stimulate the gravity receptor organs, which was likely responsible for the improved balance performance relative to strains with absent otoconia. Furthermore, the combination of direct electrophysiological measures and a series of behavioral tests can be used to interpret the imbalance severity arising from altered inputs from the gravity receptor end organ.

Calcium microdomains at presynaptic active zones of vertebrate hair cells unmasked by stochastic deconvolution

Signal transduction by auditory and vestibular hair cells involves an impressive ensemble of finely tuned control mechanisms, strictly dependent on the local intracellular Ca(2+) concentration ([Ca(2+)](i)). The study of Ca(2+) dynamics in hair cells typically combines Ca(2+)-sensitive fluorescent indicators (dyes), patch clamp and optical microscopy to produce images of the patterns of fluorescence of a Ca(2+) indicator following various stimulation protocols. Here we describe a novel method that combines electrophysiological recordings, fluorescence imaging and numerical simulations to effectively deconvolve Ca(2+) signals within cytoplasmic microdomains that would otherwise remain inaccessible to direct observation. The method relies on the comparison of experimental data with virtual signals derived from a Monte Carlo reaction-diffusion model based on a realistic reconstruction of the relevant cell boundaries in three dimensions. The model comprises Ca(2+) entry at individual presynaptic active zones followed by diffusion, buffering, extrusion and release of Ca(2+). Our results indicate that changes of the hair cell [Ca(2+)](i) during synaptic transmission are primarily controlled by the Ca(2+) endogenous buffers both at short (<1mu) and at long (tens of microns) distances from the active zones. We provide quantitative estimates of concentration and kinetics of the hair cell endogenous Ca(2+) buffers and Ca(2+)-ATPases. We finally show that experimental fluorescence data collected during Ca(2+) influx are not interpreted correctly if the [Ca(2+)](i) is estimated by assuming that Ca(2+) equilibrates instantly with its reactants. In our opinion, this approach is of potentially general interest as it can be easily adapted to the study of Ca(2+) dynamics in diverse biological systems.

Ionic composition of endolymph and perilymph in the inner ear of the oyster toadfish, Opsanus tau.

The concentrations of free Na+, K+, Ca(+, and Cl(-)in endolymph and perilymph from the inner ear of the oyster toadfish, Opsanus tau, were measured in vivo using double-barreled ion-selective electrodes. Perilymph concentrations were similar to those measured in other species, while endolymph concentrations were similar to those measured previously in elasmobranch fish, though significantly different from concentrations reported in mammals. Perilymph concentrations (mean +/- std. dev.) were as follows: Na+, 129 mmol l(-1) +/- 20; K+, 4.96 mmol l(-1) +/- 2.67; Ca2+, 1.83 mmol l(-1) +/- 0.27; and Cl(-), 171 mmol l(-1) +/- 20. Saccular endolymph concentrations were Na+, 166 mmol l(-1) +/- 22; K+, 51.4 mmol l(-1) +/- 16.7; Ca2+, 2.88 mmol l(-1) +/- 0.27; and Cl(-), 170 mmol l(-1) +/- 12; and semicircular canal (utricular vestibule) endolymph concentrations were Na+, 122 mmol l(-1) +/- 15; K+, 47.7 mmol l(-1) +/- 13.2; Ca2+, 1.78 mmol l(-1) +/- 0.48; Cl(-), 176 mmol l(-1) +/- 27. The relatively high concentrations of Ca2+ and Na+ in the endolymph may have significant implications for the physiological function of the mechanoelectrical transduction channels in the vestibular hair cells of fish compared to those of their mammalian counterparts.

Kvβ1.1 associates with Kvα1.4 in chinese hamster ovary cells and pigeon type II vestibular hair cells and enhances the amplitude, inactivation and negatively shifts the steady-state inactivation range

Although A-type potassium currents are found in type II hair cells in the inner ear of most species, the molecular mechanisms for activation and inactivation of the A-type potassium current (I A) remain unknown. In frog semicircular canal hair cells, for example, there appear to be two classes of currents having either fast or slow inactivation [Norris CH, Ricci AJ, Housley GD, Guth PS (1992) The inactivating potassium currents of hair cells isolated from the crista ampullaris of the frog. J Neurophysiol 68:1642–1653; Russo G, Calzi D, Martini M, Rossi ML, Fesce R, Prigioni I (2007) Potassium currents in the hair cells of vestibular epithelium: position-dependent expression of two types of A channels. Eur J Neurosci 25:695–704]. It has been suggested that somehow the “ball and chain” mechanism (NH 3 (N) terminus motif) is modified by alternative splicing to account for the two classes of inactivation. To examine other possibilities, we cloned alpha and beta subunits that comprise the A-type potassium channel complex in adult and embryonic pigeon brain, cochlea and labyrinth. By sequence homology, we concluded that the subunits present were Kvα1.4 and Kvβ1.1. The sequence of the open reading frame for Kvα1.4 contained the N-terminus, pore and COOH (C) terminus motifs for N-and C-type inactivation. The sequence for Kvβ1.1 displayed amino acids consistent with assembly and association with Kvα1.4 alpha subunits. Kvα1.4 and Kvβ1.1 were transfected either singly or in combination into Chinese hamster ovary (CHO) cells. These cells and native hair cells from the pigeon utricle were patch clamped and the inactivation properties of the A-type current were studied. In the native hair cells, the A-type current was identified by its pharmacological (4-aminopyridine (4-AP); IC 50=11 μM) and voltage dependent inactivation properties. A comparison of the mean time constants from best-fitted single exponential and sum of two exponential equations to the ionic current inactivation revealed the following. In CHO cells when Kvα1.4 was expressed alone, the mean time constant (τ 1=107 ms±19, N=32) was significantly ( P<0.001) longer and the mean peak amplitude (2.28 nA±0.39, N=32) was smaller than when Kvα1.4 and Kvβ1.1 were expressed in CHO cells. Moreover, the co-transfection of Kvα1.4 and Kvβ1.1 into CHO cells caused a shift in the steady state inactivation curve parameter Vo 30 mV in the hyperpolarized direction relative to CHO cells expressing only Kvα1.4. Similarly, Kvα1.4-transfected CHO cells produced longer time constants and smaller amplitudes than those found for native utricular hair cells. These data lead us to conclude that while the amino acid motifs are present in Kvα1.4 and Kvβ1.1 to suggest N-and C-type inactivation, co-assembly and association of Kvα1.4 and Kvβ1.1 may also produce changes in the time dependent inactivation properties of vestibular hair cells.

Effects of hydrogen peroxide on vestibular hair cells in the guinea pig: importance of cell membrane impairment preceding cell death

Conclusion. Our findings indicate that oxidative stress induces morphological changes in vestibular hair cells and subsequently leads to cell death after 2.5 h. Objectives. The aim of this study was to confirm the direct effects of oxidative stress on vestibular hair cells. Materials and methods. Vestibular hair cells isolated from guinea pigs were loaded with 1 or 10 mM H2O2, and morphological changes were observed. In addition, in a viability/cytotoxicity assay system, the numbers of dead cells in isolated cristae ampullares were counted 1, 3, and 5 h after loading with H2O2 or artificial perilymph (control). Results. Reactive oxygen, in the form of H2O2, directly affects the cell membrane of isolated vestibular hair cells and causes swelling of the cell body, bleb formation, and shortening of the neck region. Morphological changes occur within 30 min after loading with H2O2, but a significant increase in the number of dead cells is noted only after 3 h.

Molecular modelling insights into DFNA15 mediated enhancement of POU4F3 stability

The POU4F3 transcription factor is expressed in the cochlear and vestibular hair cells of the inner ear and its targeted deletion results in a loss of inner ear hair cells. The DFNA15 truncation mutation has been demonstrated to result in a loss of transcriptional activity, but an increase in the stability of the protein. Molecular modelling is utilised to propose a mechanism of stability enhancement, via an interaction between the truncated POU(HD) domain and the POU(S) domain of the transcription factor.

Sox2 and Jagged1 Expression in Normal and Drug-Damaged Adult Mouse Inner Ear

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.