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Intratympanic injection of gentamicin has proven to be an effective therapy for intractable vestibular dysfunction. However, most studies to date have focused on the cochlea, so little is known about the distribution and uptake of gentamicin by the counterpart of the auditory system, specifically vestibular hair cells (HCs). Here, with a combination of in vivo and in vitro approaches, we used a gentamicin-Texas Red (GTTR) conjugate to investigate the mechanisms of gentamicin vestibulotoxicity in the developing mammalian utricular HCs. In vivo, GTTR fluorescence was concentrated in the apical cytoplasm and the cellular membrane of neonatal utricular HCs, but scarce in the nucleus of HCs and supporting cells. Quantitative analysis showed the GTTR uptake by striolar HCs was significantly higher than that in the extrastriola. In addition, the GTTR fluorescence intensity in the striola was increased gradually from 1 to 8 days, peaking at 8-9 days postnatally. In vitro, utricle explants were incubated with GTTR and candidate uptake conduits, including mechanotransduction (MET) channels and endocytosis in the HC, were inhibited separately. GTTR uptake by HCs could be inhibited by quinine, a blocker of MET channels, under both normal and stressed conditions. Meanwhile, endocytic inhibition only reduced GTTR uptake in the CoCl2 hypoxia model. In sum, the maturation of MET channels mediated uptake of GTTR into vestibular HCs. Under stressed conditions, MET channels play a pronounced role, manifested by channel-dependent stress enhanced GTTR permeation, while endocytosis participates in GTTR entry in a more selective manner.

To explore the effects of cochlear hair cell displacement, researchers have previously monitored functional and mechanical responses during low-frequency (LF) acoustic stimulation of the cochlea. The induced changes are believed to result from modulation of the conductance of mechano-electrical transduction (MET) channels on cochlear hair cells, along with receptor potential modulation. It is less clear how, or if, vestibular hair cell displacement affects vestibular function. Here, we have used LF (

Hair cells are the sensory receptors in the auditory and vestibular systems of all vertebrates, and in the lateral-line system of aquatic vertebrates. During development, spontaneous activity in hair cells shapes the formation of these sensory systems. In auditory hair cells of mice, coordinated waves of spontaneous activity can be triggered by concomitant activity in nearby supporting cells. But in mammals, developing auditory and vestibular hair cells can also autonomously generate spontaneous events independent of supporting cell activity. To date, significant progress has been made studying spontaneous activity in the auditory and vestibular systems of mammals, in isolated cultures. The purpose of this work is to explore the zebrafish lateral-line system as a model to study and understand spontaneous activity in vivo. Our work applies genetically encoded calcium indicators along with light-sheet fluorescence microscopy to visualize spontaneous calcium activity in the developing lateral-line system. Consistent with our previous work, we show that spontaneous calcium activity is present in developing lateral-line hair cells. We now show that supporting cells that surround hair cells, and cholinergic efferent terminals that directly contact hair cells are also spontaneously active. Using two-color functional imaging we demonstrate that spontaneous activity in hair cells does not correlate with activity in either supporting cells or cholinergic terminals. We find that during lateral-line development, hair cells autonomously generate spontaneous events. Using localized calcium indicators, we show that within hair cells, spontaneous calcium activity occurs in two distinct domains–the mechanosensory bundle and the presynapse. Further, spontaneous activity in the mechanosensory bundle ultimately drives spontaneous calcium influx at the presynapse. Comprehensively, our results indicate that in developing lateral-line hair cells, autonomously generated spontaneous activity originates with spontaneous mechanosensory events. Overall, with robust spontaneous activity three different cell types, the developing lateral line is a rich model to study these activities in an intact sensory organ. Future work studying this model may further our understanding of these activities and their role in sensory system formation, function and regeneration.

Vestibular hair cells transmit information about head position and motion across synapses to primary afferent neurons. At some of these synapses, the afferent neuron envelopes the hair cell, forming an enlarged synaptic terminal called a calyx. The vestibular hair cell-calyx synapse supports a mysterious form of electrical transmission that does not involve gap junctions termed nonquantal transmission (NQT). The NQT mechanism is thought to involve the flow of ions from the pre-synaptic hair cell to the post-synaptic calyx through low-voltage-activated channels driven by changes in cleft [K+] as K+ exits the hair cell. However, this hypothesis has not been tested with a quantitative model and the possible role of an electrical potential in the cleft has remained speculative. Here we present a computational model that captures salient experimental observations of NQT and identifies overlooked features that corroborate the existence of an electrical potential (ϕ) in the synaptic cleft. We show that changes in cleft ϕ reduce transmission latency and illustrate the relative contributions of both cleft [K+] and ϕ to the gain and phase of NQT. We further demonstrate that the magnitude and speed of NQT depend on calyx morphology and that increasing calyx height reduces action potential latency in the calyx afferent. These predictions are consistent with the idea that the calyx evolved to enhance NQT and speed up vestibular signals that drive neural circuits controlling gaze, balance, and orientation.Significance StatementThe ability of the vestibular system to drive the fastest reflexes in the nervous system depends on rapid transmission of mechanosensory signals at vestibular hair cell synapses. In mammals and other amniotes, afferent neurons form unusually large calyx terminals on certain hair cells, and communication at these synapses includes nonquantal transmission (NQT), which avoids the synaptic delay of quantal transmission. We present a quantitative model that shows how NQT depends on the extent of the calyx covering the hair cell and attributes the short latency of NQT to changes in synaptic cleft electrical potential caused by current flowing through open potassium channels in the hair cell. This previously undescribed mechanism may act at other synapses.

A previous study showed that people living in urban areas are generally exposed to low-frequency noise (LFN) with frequencies below 100 Hz and sound levels of 60–110 dB in daily and occupational environments. Exposure to LFN has been shown to affect balance in humans and mice. However, there is no information about prevention of LFN-mediated imbalance because of a lack of information about the target region based on health risk assessment of LFN exposure. Here, we show that acute exposure to LFN at 100 Hz, 95 dB, but not at 85 dB or 90 dB, for only 1 h caused imbalance in mice. The exposed mice also had decreased cervical vestibular-evoked myogenic potential (cVEMP) with impaired activity of vestibular hair cells. Since imbalance in the exposed mice was irreversible, morphological damage in the vestibules of the exposed mice was further examined. The exposed mice had breakage of the otoconial membrane in the vestibule. LFN-mediated imbalance and breakage of the otoconial membrane in mice were rescued by overexpression of a stress-reactive molecular chaperone, heat shock protein 70 (Hsp70), which has been shown to be induced by exposure of mice to 12 h per day of LFN at 95 dB for 5 days. Taken together, the results of this study demonstrate that acute exposure to LFN at 100 Hz, 95 dB for only 1 h caused irreversible imbalance in mice with structural damage of the otoconial membrane as the target region for LFN-mediated imbalance, which can be rescued by Hsp70.

To better understand the spread of prosthetic current in the inner ear and to facilitate design of electrode arrays and stimulation protocols for a vestibular implant system intended to restore sensation after loss of vestibular hair cell function, we created a model of the primate labyrinth. Because the geometry of the implanted ear is complex, accurately modeling effects of prosthetic stimuli on vestibular afferent activity required a detailed representation of labyrinthine anatomy. Model geometry was therefore generated from three-dimensional (3D) reconstructions of a normal rhesus temporal bone imaged using micro-MRI and micro-CT. For systematically varied combinations of active and return electrode location, the extracellular potential field during a biphasic current pulse was computed using finite element methods. Potential field values served as inputs to stochastic, nonlinear dynamic models for each of 2415 vestibular afferent axons, each with unique origin on the neuroepithelium and spiking dynamics based on a modified Smith and Goldberg model. We tested the model by comparing predicted and actual 3D vestibulo-ocular reflex (VOR) responses for eye rotation elicited by prosthetic stimuli. The model was individualized for each implanted animal by placing model electrodes in the standard labyrinth geometry based on CT localization of actual implanted electrodes. Eye rotation 3D axes were predicted from relative proportions of model axons excited within each of the three ampullary nerves, and predictions were compared to archival eye movement response data measured in three alert rhesus monkeys using 3D scleral coil oculography. Multiple empirically observed features emerged as properties of the model, including effects of changing active and return electrode position. The model predicts improved prosthesis performance when the reference electrode is in the labyrinth's common crus (CC) rather than outside the temporal bone, especially if the reference electrode is inserted nearly to the junction of the CC with the vestibule. Extension of the model to human anatomy should facilitate optimal design of electrode arrays for clinical application.

Objectives: Early in his career, David Lim recognized the scientific impact of genetically anomalous mice exhibiting otoconia agenesis as models of drastically compromised vestibular function. While these studies focused on the mutant pallid mouse, contemporary genetic tools have produced other models with engineered functional modifications. Lim and colleagues foresaw the need to analyze vestibular epithelia from pallid mice to verify the absence of downstream consequences that might be secondary to the altered load represented by otoconial agenesis. More generally, however, such comparisons also contribute to an understanding of the susceptibility of labyrinthine sensory epithelia to more widespread cellular changes associated with what may appear as isolated modifications. Methods: Our laboratory utilizes a model of vestibular hypofunction produced through genetic alteration, the otoferlin-null mouse, which has been shown to exhibit severely compromised stimulus-evoked neurotransmitter release in type I hair cells of the utricular striola. The present study, reminiscent of early investigations of Lim and colleagues that explored the utility of a genetically altered mouse to explore its utility as a model of vestibular hypofunction, endeavored to compare the expression of the hair cell marker oncomodulin in vestibular epithelia from wild-type and otoferlin-null mice. Results: We found that levels of oncomodulin expression were much greater in type I than type II hair cells, though were similar across the 3 genotypes examined (ie, including heterozygotes). Conclusion: These findings support the notion that modifications resulting in a specific component of vestibular hypofunction are not accompanied by widespread morphologic and cellular changes in the vestibular sensory epithelia.

In superior canal dehiscence (SCD), fluid displacement of the endolymph activates type I vestibular hair cells in the crista of the affected canal and thus irregular superior canal (SC) neurons in Scarpa's ganglion, which provides the neurophysiological basis for the clinical presentation of SCD.Patients with SCD display sound- and vibration-induced vertigo/nystagmus and increased amplitudes of vestibular evoked myogenic potentials.Extracellular recordings from n = 25 primary vestibular neurons of 16 female guinea pigs were analyzed. We recorded from the same vestibular neuron before, during and after creating the dehiscence and after closing the dehiscence. Neurobiotin labeling was employed in n = 11 neurons.After SCD, previously unresponsive irregular SC neurons displayed a stimulus-locked increase in discharge during application of air-conducted sound (ACS) or bone-conducted vibration (BCV) for a broad range of frequencies (ACS: 200-4000 Hz; BCV: 500-1500 Hz). This typical response was only observed for irregular SC neurons (n = 19), but not regular SC neurons, or irregular/regular horizontal canal neurons (n = 2 each), and was abolished after closing the dehiscence. Eleven irregular SC neurons responsive to ACS and/or BCV were traced back to calyx synapses in the central crista of the affected superior canal by neurobiotin labeling.Stimulus-locked activation of irregular SC neurons by ACS and BCV is the neurophysiological basis for sound- and vibration-induced vertigo/nystagmus and increased VEMP amplitudes in SCD. The results of the present study help to improve vestibular diagnostics in patients with suspected SCD.

The vestibular organs of reptiles, birds, and mammals possess type I and type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a type I or type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse utricles between postnatal day 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to type I hair cells) increased in all regions of the utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER(T2):Rosa26(tdTomato) mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were type II. Our findings show that most neonatally-derived hair cells become type II, and many type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.

Drug delivery to the inner ear by nanomedicine strategies has emerged as an effective therapeutic approach for the management of inner ear diseases including hearing and balance disorders. It is well accepted that substance enters the perilymph from the middle ear through the round window membrane (RWM), but the passage through the oval window (OW) has long been neglected. Up to now, researchers still know little about the pathway via which nanoparticles (NPs) enter the inner ear or how they reach the inner ear following local applications. Herein, we engineered fluorescence traceable chitosan (CS) NPs, investigated the NP distribution within cochlear and vestibular organs, and assessed the availability of RWM and OW pathways to NP transport. Intriguingly, there were high levels of CS NPs in vestibular hair cells, dark cells and supporting cells, but negligible ones in cochlear hair cells and epithelial cells after intratympanic administration. However, the NPs were visualized in two cell models, L929 and HEI-OC1 cell lines, and in the hair cells of cochlear explants after co-incubation in vitro. These combined studies implied that CS NPs might enter the vestibule directly through the OW and then preferentially accumulated in the cells of vestibular organs. Thus, in vivo studies were carried out and clearly revealed that CS NPs entered the inner ear through both the RWM and OW, but the latter played a governing role in delivering NPs to the vestibule with vivid fluorescence signals in the thin bone of the stapes footplate. Overall, these findings firstly suggested that the OW, as a royal gate, afforded a convenient access to facilitate CS NPs transport into inner ear, casting a new light on future clinical applications of NPs in the effective treatment of vestibular disorders by minimizing the risk of hearing loss associated with cochlear hair cell pathology.

In this work, an ultra-sensitive flow sensor is presented, consisting of vertically grown graphene nanosheets (VGNs) with a mazelike structure and an elastomer (polydimethylsiloxane, PDMS). The VGNs/PDMS piezoresistive flow sensor exhibits great linearity, low-velocity detection threshold (1.127 mm/s) and super-high sensitivity under exposure to stationary flow (0.127 kΩ/(mL/min)). The proposed flow sensor, analogous to hair cells in the vestibular system, was embedded in a 3D-printed lateral semicircular canal, and the sensing performance was studied in response to various physiological movements. This work paves the way for development of physical sensors using novel two-dimensional (2D) materials for various biomedical applications.

Annexin A4 (ANXA4) is a Ca2+-dependent phospholipid-binding protein that is specifically expressed in the cochlear and vestibular hair cells, but its function in the hair cells remains unknown. In the present study, we show that besides localizing on the plasma membrane, ANXA4 immunoreactivity is also localized at the tips of stereocilia in the hair cells. In order to investigate the role of ANXA4 in the hair cells, we established Anxa4 knockout mice using CRISPR/Cas9 technique. Unexpectedly, the development of both cochlear and vestibular hair cells is normal in Anxa4 knockout mice. Moreover, stereocilia morphology of Anxa4 knockout mice is normal, so is the mechano-electrical transduction (MET) function. Consistently, the auditory and vestibular functions are normal in the knockout mice. In conclusion, we show here that ANXA4 is dispensable for the development and function of hair cells, which might result from functional redundancy between ANXA4 and other annexin(s) in the hair cells.

Humans rely on multiple senses to perceive their self-motion in the real world. For example, a sideways linear head translation can be sensed either by lamellar optic flow of the visual scene projected on the retina of the eye or by stimulation of vestibular hair cell receptors found in the otolith macula of the inner ear. Mismatches in visual and vestibular information can induce cybersickness during head-mounted display (HMD) based virtual reality (VR). In this pilot study, participants were immersed in a virtual environment using two recent consumer-grade HMDs: the Oculus Go (3DOF angular only head tracking) and the Oculus Quest (6DOF angular and linear head tracking). On each trial they generated horizontal linear head oscillations along the interaural axis at a rate of 0.5 Hz. This head movement should generate greater sensory conflict when viewing the virtual environment on the Oculus Go (compared to the Quest) due to the absence of linear tracking. We found that perceived scene instability always increased with the degree of linear visual-vestibular conflict. However, cybersickness was not experienced by 7/14 participants, but was experienced by the remaining participants in at least one of the stereoscopic viewing conditions (six of whom also reported cybersickness in monoscopic viewing conditions). No statistical difference in spatial presence was found across conditions, suggesting that participants could tolerate considerable scene instability while retaining the feeling of being there in the virtual environment. Levels of perceived scene instability, spatial presence and cybersickness were found to be similar between the Oculus Go and the Oculus Quest with linear tracking disabled. The limited effect of linear coupling on cybersickness, compared with its strong effect on perceived scene instability, suggests that perceived scene instability may not always be associated with cybersickness. However, perceived scene instability does appear to provide explanatory power over the cybersickness observed in stereoscopic viewing conditions.

Vestibular hair cells are mechanosensory receptors that are capable of detecting changes in head position and thereby allow animals to maintain their posture and coordinate their movement. Vestibular hair cells are susceptible to ototoxic drugs, aging, and genetic factors that can lead to permanent vestibular dysfunction. Vestibular dysfunction mainly results from the injury of hair cells, which are located in the vestibular sensory epithelium. This review summarizes the mechanisms of different factors causing vestibular hair cell damage and therapeutic strategies to protect vestibular hair cells.

The Summating Potential (SP) was first recorded in the cochlea in the 1950s and represents an objective measure of cochlear hair cell function, in vivo. Despite being a regular tool in hearing research, a similar response has not yet been recorded from the vestibular system. This is mainly due to the lack of experimental techniques available to record electrical vestibular hair cell responses in isolation from the much larger cochlear potentials. Here we demonstrate the first recordings of the vestibular SP, evoked by Bone-Conducted Vibration (BCV) and Air-Conducted Sound (ACS) stimuli, in anaesthetized guinea pigs. Field potential measurements were taken from the basal surface of the utricular macula, and from the facial nerve canal following surgical or chemical ablation of the cochlea. SPs were evoked by stimuli with frequencies above ~200 Hz, and only with moderate to high intensity (~0.005–0.05 g) BCV and ACS (~120–140 dB SPL). Neural blockade abolished the Vestibular short-latency Evoked Potential (VsEP) and Vestibular Nerve Neurophonic (VNN) from the facial nerve canal recordings but did not abolish the vestibular SP nor the vestibular microphonic. Importantly, the vestibular SP was irreversibly abolished from the utricle and facial nerve canal recordings following local gentamicin application, highlighting its hair cell origin. This is the first study to record the Summating Potential from the mammalian vestibular system, in vivo, providing a novel research tool to assess vestibular hair cell function during experimental manipulations and animal models of disease.

The tail-lift reflex and the air-righting reflex in rats are anti-gravity reflexes that depend on vestibular function. To begin identifying their cellular basis, this study examined the relationship between reflex loss and the graded lesions caused in the vestibular sensory epithelia by varying doses of an ototoxic compound. After ototoxic exposure, we recorded these reflexes using high speed video. The movies were used to obtain objective measures of the reflexes: the minimum angle formed by the nose, the back of the neck and the base of the tail during the tail-lift maneuver and the time to right in the air-righting test. The vestibular sensory epithelia were then collected from the rats and used to estimate the loss of type I (HCI), type II (HCII) and all hair cells (HC) in both central and peripheral parts of the crista, utricle, and saccule. As expected, tail-lift angles decreased, and air-righting times increased, while the numbers of HCs remaining in the epithelia decreased in a dose-dependent manner. The results demonstrated greater sensitivity of HCI compared to HCII to the IDPN ototoxicity, as well as a relative resiliency of the saccule compared to the crista and utricle. Comparing the functional measures with the cell counts, we observed that loss of the tail-lift reflex associates better with HCI than with HCII loss. In contrast, most HCI in the crista and utricle were lost before air-righting times increased. These data suggest that these reflexes depend on the function of non-identical populations of vestibular HCs.

The vestibular system can be thought of as a system that senses and controls motion. Its functions begin with the detection of head position and motion by the vestibular end-organs. The vestibular hair cells transduce the mechanical stimuli into neural signals and convey them to the brainstem. The brainstem processes the signals and distributes it to other areas of the central nervous system (CNS) to generate vestibular reflexes and sensation. Vestibular reflexes stabilize both our posture and our gait and have significant effects on our tests of vestibular function. In this chapter, we will discuss the physiology of the vestibular system and the different tools used to assess the integrity of the vestibular system.

Noisy galvanic vestibular stimulation (nGVS) is an emerging non-invasive brain stimulation technique. It involves applying alternating currents of different frequencies and amplitudes presented in a random, or noisy, manner through electrodes on the mastoid bones behind the ears. Because it directly activates vestibular hair cells and afferents and has an indirect effect on a variety of brain regions, it has the potential to impact many different functions. The objective of this review is twofold: (1) to review how nGVS affects motor, sensory, and cognitive performance in healthy adults; and (2) to discuss potential clinical applications of nGVS. First, we introduce the technique. We then describe the regions receiving and processing vestibular information. Next, we discuss the effects of nGVS on motor, sensory, and cognitive function in healthy adults. Subsequently, we outline its potential clinical applications. Finally, we highlight other electrical stimulation technologies and discuss why nGVS offers an alternative or complementary approach. Overall, nGVS appears promising for optimizing human performance and as an assistive technology, though further research is required.

Decades-old model of slow adaptation in sensory hair cells of the auditory and vestibular systems requires revamp., Hair cells detect sound and motion through a mechano-electric transduction (MET) process mediated by tip links connecting shorter stereocilia to adjacent taller stereocilia. Adaptation is a key feature of MET that regulates a cell’s dynamic range and frequency selectivity. A decades-old hypothesis proposes that slow adaptation requires myosin motors to modulate the tip-link position on taller stereocilia. This “motor model” depended on data suggesting that the receptor current decay had a time course similar to that of hair-bundle creep (a continued movement in the direction of a step-like force stimulus). Using cochlear and vestibular hair cells of mice, rats, and gerbils, we assessed how modulating adaptation affected hair-bundle creep. Our results are consistent with slow adaptation requiring myosin motors. However, the hair-bundle creep and slow adaptation were uncorrelated, challenging a critical piece of evidence upholding the motor model. Considering these data, we propose a revised model of hair cell adaptation.

The sense of balance relies on vestibular hair cells, which detect head motions. Mammals have two types of vestibular hair cell, I and II, with unique morphological, molecular, and physiological properties. Furthermore, each hair cell type synapses on a unique form of afferent nerve terminal. Little is known about the mechanisms in mature animals that maintain the specific features of each hair cell type or its post-synaptic innervation. We found that deletion of the transcription factor Sox2 from type II hair cells in adult mice of both sexes caused many cells in utricles to acquire features unique to type I hair cells and to lose type II-specific features. This cellular transdifferentiation, which included changes in nuclear size, chromatin condensation, soma and stereocilium morphology, and marker expression, resulted in a significantly higher proportion of type I-like hair cells in all epithelial zones. Furthermore, Sox2 deletion from type II hair cells triggered non-cell autonomous changes in vestibular afferent neurons; they retracted bouton terminals (normally present on only type II cells) from transdifferentiating hair cells and replaced them with a calyx terminal (normally present on only type I cells). These changes were accompanied by significant expansion of the utricle's central zone, called the striola. Our study presents the first example of a transcription factor required to maintain the type-specific hair cell phenotype in adult inner ears. Furthermore, we demonstrate that a single genetic change in type II hair cells is sufficient to alter the morphology of their post-synaptic partners, the vestibular afferent neurons.SIGNIFICANCE STATEMENT:The sense of balance relies on two types of sensory cells in the inner ear - type I and type II hair cells. These two cell types have unique properties. Furthermore, their post-synaptic partners, the vestibular afferent neurons, have differently shaped terminals on type I versus type II hair cells. We show that the transcription factor Sox2 is required to maintain the cell-specific features of type II hair cells and their post-synaptic terminals in adult mice. This is the first evidence of a molecule that maintains the phenotypes of hair cells and, non-cell autonomously, their post-synaptic partners in mature animals.

In the vestibular peripheral organs, type I and type II hair cells (HCs) transmit incoming signals via glutamatergic quantal transmission onto afferent nerve fibers. Additionally, type I HCs transmit via ‘non-quantal’ transmission to calyx afferent fibers, by accumulation of glutamate and potassium in the synaptic cleft. Vestibular efferent inputs originating in the brainstem contact type II HCs and vestibular afferents. Here, we aimed at characterizing the synaptic efferent inputs to type II HCs using electrical and optogenetic stimulation of efferent fibers combined with in vitro whole-cell patch clamp recording from type II HCs in the rodent vestibular crista. Properties of efferent synaptic currents in type II HCs were similar to those found in cochlear hair cells and mediated by activation of α9/α10 nicotinic acetylcholine receptors (AChRs) and SK potassium channels. While efferents showed a low probability of release at low frequencies of stimulation, repetitive stimulation resulted in facilitation and increased probability of release. Notably, the membrane potential of type II HCs measured during optogenetic stimulation of efferents showed a strong hyperpolarization even in response to single pulses and was further enhanced by repetitive stimulation. Such efferent-mediated inhibition of type II HCs can provide a mechanism to adjust the contribution of signals from type I and type II HCs to vestibular nerve fibers. As a result, the relative input of type I hair cells to vestibular afferents will be strengthened, emphasizing the phasic properties of the incoming signal that are transmitted via fast non-quantal transmission.New and NoteworthyType II vestibular hair cells (HCs) receive inputs from efferent fibers originating in the brainstem. We used in vitro optogenetic and electrical stimulation of efferent fibers to study their synaptic inputs to type II HCs. Efferent inputs inhibited type II HCs, similar to cochlear efferent effects. We propose that efferent inputs adjust the contribution of signals from type I and type II HCs that report different components of the incoming signal to vestibular nerve fibers.

Mature hair cells transduce information over a wide range of stimulus intensities and frequencies for prolonged periods of time. The efficiency of such a demanding task is reflected in the characteristics of exocytosis at their specialized presynaptic ribbons. Ribbons are electron‐dense structures able to tether a large number of releasable vesicles allowing them to maintain high rates of vesicle release. Calcium entry through rapidly activating, non‐inactivating CaV1.3 (L‐type) Ca2+ channels in response to cell depolarization causes a local increase in Ca2+ at the ribbon synapses, which is detected by the exocytotic Ca2+ sensors. The Ca2+ dependence of vesicle exocytosis at mammalian vestibular hair cell (VHC) ribbon synapses is believed to be linear, similar to that observed in mature cochlear inner hair cells (IHCs). The linear relation has been shown to correlate with the presence of the Ca2+ sensor synaptotagmin‐4 (Syt‐4). Therefore, we studied the exocytotic Ca2+ dependence, and the release kinetics of different vesicle pool populations, in Type II VHCs of control and Syt‐4 knockout mice using patch‐clamp capacitance measurements, under physiological recording conditions. We found that exocytosis in mature control and knockout Type II VHCs displayed a high‐order dependence on Ca2+ entry, rather than the linear relation previously observed. Consistent with this finding, the Ca2+ dependence and release kinetics of the ready releasable pool (RRP) of vesicles were not affected by an absence of Syt‐4. However, we did find that Syt‐4 could play a role in regulating the release of the secondary releasable pool (SRP) in these cells. Our findings show that the coupling between Ca2+ influx and neurotransmitter release at mature Type II VHC ribbon synapses is faithfully described by a nonlinear relation that is likely to be more appropriate for the accurate encoding of low‐frequency vestibular information, consistent with that observed at low‐frequency mammalian auditory receptors., Here we show that the coupling between neurotransmitter release at the ribbon synapses of mature vestibular Type II hair cells display a high‐order dependence on calcium influx. The calcium dependence was not affected by an absence of the calcium‐sensing synaptic molecule synaptotagmin‐4, which has been shown to be involved in establishing the linear calcium dependence of high‐frequency auditory hair cells. Our findings suggest that a nonlinear exocytotic calcium dependence in vestibular hair cells is likely to be more appropriate for the accurate encoding of low‐frequency vestibular information.

Loss of hair cells from vestibular epithelium results in balance dysfunction. The current therapeutic regimen for vestibular diseases is limited. Upon injury or Atoh1 overexpression, hair cell replacement occurs rapidly in the mammalian utricle, suggesting a promising approach to induce vestibular hair cell regeneration. In this study, we applied simultaneous gentamicin-mediated hair cell ablation and Atoh1 overexpression to induce neonatal utricular hair cell formation in vitro. We confirmed that type I hair cells were the primary targets of gentamicin. Furthermore, injury and Atoh1 overexpression promoted hair cell regeneration in a timely and efficient manner through robust viral transfection. Hair cells regenerated with type II characteristics in the striola and type I/II characteristics in non-sensory regions. Rare EdU+/myosin7a+ cells in sensory regions and robust EdU+/myosin7a+ signals in ectopic regions indicate that transdifferentiation of supporting cells in situ, and mitosis and differentiation of non-sensory epithelial cells in ectopic regions, are sources of regenerative hair cells. Distinct regeneration patterns in in situ and ectopic regions suggested robust plasticity of vestibular non-sensory epithelium, generating more developed hair cell subtypes and thus providing a promising stem cell-like source of hair cells. These findings suggest that simultaneously causing injury and overexpressing Atoh1 promotes hair cell regeneration efficacy and maturity, thus expanding the understanding of ectopic plasticity in neonatal vestibular organs.

Background Vestibular hair cell loss and its role in balance disorders are not yet completely understood due largely to the lack of precise hair cell damage protocols. New method Our damage protocol aims to selectively remove type I hair cells in a way that produces consistent and predictable lesions that can be used for reliable inter-animal and inter-group comparison in balance research. This objective is achieved by transtympanic injection of gentamicin on both the round window membrane and oval window over a fixed time period followed by thorough washing. Results We achieved nearly total and consistent loss of type I hair cells at 94 % for the crista ampullaris of the lateral semicircular canal (LSC) and 86 % for the utricular macula with negligible loss of type II hair cells at 4% for the crista ampullaris of the LSC and 6% for the utricular macula. While the vestibular function was compromised in the relevant study group, this group had a zero mortality rate with no significant suppression of body weight gain. Comparison with existing methods Gentamicin is typically administered via intraperitoneal systemic injection or, more recently, transtympanic injection. The intraperitoneal method is simple, but mortality rate is high. The transtympanic injection method produces ototoxic damage but with inconsistent lesion size. This inconsistency prevents reliable comparisons among animals. Conclusions This protocol employs a transtympanic injection method which selectively targets type I hair cells for removal in the vestibular epithelia in a time-dependent manner, uniformly damages vestibular function, and causes uniform hair cell loss.

Hair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed. Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.

FGF8 signaling plays diverse roles in inner ear development, acting at multiple stages from otic placode induction to cellular differentiation in the organ of Corti. As a secreted morphogen with diverse functions, Fgf8 expression is likely to be spatially restricted and temporally dynamic throughout inner ear development. We evaluated these characteristics using genetic labeling mediated by Fgf8mcm gene-targeted mice and determined that Fgf8 expression is a specific and early marker of Type-I vestibular hair cell identity. Fgf8mcm expression initiates at E11.5 in the future striolar region of the utricle, labeling hair cells following EdU birthdating, and demonstrates that sub-type identity is determined shortly after terminal mitosis. This early fate specification is not apparent using markers or morphological criteria that are not present before birth in the mouse. While analyses of Fgf8 conditional knockout mice did not reveal developmental phenotypes, the restricted pattern of Fgf8 expression suggests that functionally redundant FGF ligands may contribute to vestibular hair cell differentiation and supports a developmental model in which Type-I and Type-II hair cells develop in parallel rather than from an intermediate precursor.

Objective To investigate the temporal bone histopathology of vasculitis, especially in the vestibular organs, in granulomatosis with polyangitis (GPA). Methods Using light and differential interference contrast microscopy, we examined 12 human temporal bones from six deceased GPA patients and 12 histopathologically normal human temporal bones from six deceased age-matched patients. Results In the GPA group, three patients had undergone tympanostomy tube placement. Two of them had suffered mixed hearing loss; one, sensorineural hearing loss; and one, conductive hearing loss. Of the 12 specimens in the GPA group, the granulation tissue invaded the round window niche in seven; cochlear hair cells were not preserved in five. Hemosiderin was deposited in the stria vascularis in eight specimens, in the ampulla or semicircular duct in 10, and in the vestibule in three. The spiral ligament showed severe loss of cellularity in two specimens. In the GPA group, type I vestibular hair cell density was significantly decreased; however, type II vestibular hair cell density did not significantly differ between the GPA group and the control group. Conclusion Our histopathologic findings in human temporal bone specimens of GPA patients delineated changes in the tympanic membrane, middle ear cavity, round window membrane, organ of Corti, stria vascularis, spiral ligament, ampulla, semicircular duct, and vestibule. Type I vestibular hair cell density significantly decreased in the GPA group, as compared with the control group. Level of evidence N/A.

Purpose of review The primary purpose of this review is to summarize current literature in the field of vestibular regeneration with a focus on recent developments in molecular and gene therapies. Recent findings Since the discovery of limited vestibular hair cell regeneration in mammals in the 1990s, many elegant studies have improved our knowledge of mechanisms of development and regeneration of the vestibular system. A better understanding of the developmental pathways of the vestibular organs has fueled various biological strategies to enhance regeneration, including novel techniques in deriving vestibular hair cells from embryonic and induced pluripotent stem cells. In addition, the identification of specific genetic mutations responsible for vestibular disorders has opened various opportunities for gene replacement therapy. Summary Vestibular dysfunction is a significant clinical problem with limited therapeutic options, warranting research on biological strategies to repair/regenerate the vestibular organs to restore function. The use of gene therapy appears promising in animal models of vestibular dysfunction.

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9−/−) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9−/− mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.

CIB2 is a Ca(2+)- and Mg(2+)-binding protein essential for mechanoelectrical transduction (MET) by cochlear hair cells but not by vestibular hair cells that co-express CIB2 and CIB3. Here we show that in cochlear hair cells CIB3 can functionally substitute for CIB2. Using X-ray crystallography, we demonstrate that CIB2 and CIB3 are structurally similar to KChIP proteins, auxiliary subunits of voltage-gated K(v)4 channels. CIB2 and CIB3 bind to TMC1/2 through a domain in TMC1/2 flanked by transmembrane domains 2 and 3. The co-crystal structure of the CIB-binding domain in TMC1 with CIB3 reveals that interactions are mediated through a conserved CIB hydrophobic groove, similar to KChIP1 binding of K(v)4. Functional studies in mice show that CIB2 regulates TMC1/2 localization and function in hair cells, processes that are affected by deafness-causing CIB2 mutations. We conclude that CIB2 and CIB3 are MET channel auxiliary subunits with striking similarity to K(v)4 channel auxiliary subunits.

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells. To characterize ELMOD1 control of trafficking, we analyzed mice of both sexes from a strain lacking functional ELMOD1 [roundabout (rda)]. Inrda/rdamice, cuticular plates of utricle hair cells initially formed normally, then degenerated after postnatal day 5; large numbers of vesicles invaded the compromised cuticular plate. Hair bundles initially developed normally, but the cell's apical membrane lifted away from the cuticular plate, and stereocilia elongated and fused. Membrane trafficking in type I hair cells, measured by FM1-43 dye labeling, was altered inrda/rdamice. Consistent with the proposed GAP role for ELMOD1, the ARF6 GTP/GDP ratio was significantly elevated inrda/rdautricles compared with controls, and the level of ARF6-GTP was correlated with the severity of therda/rdaphenotype. These results suggest that conversion of ARF6 to its GDP-bound form is necessary for final stabilization of the hair bundle.SIGNIFICANCE STATEMENTAssembly of the mechanically sensitive hair bundle of sensory hair cells requires growth and reorganization of apical actin and membrane structures. Hair bundles and apical membranes in mice with mutations in theElmod1gene degenerate after formation, suggesting that the ELMOD1 protein stabilizes these structures. We show that ELMOD1 is a GTPase-activating protein in hair cells for the small GTP-binding protein ARF6, known to participate in actin assembly and membrane trafficking. We propose that conversion of ARF6 into the GDP-bound form in the apical domain of hair cells is essential for stabilizing apical actin structures like the hair bundle and ensuring that the apical membrane forms appropriately around the stereocilia.

Background The Vestibular Microphonic (VM) has only featured in a handful of publications, mostly involving non-mammalian and ex vivo models. The VM is the extracellular analogue of the vestibular hair cell receptor current, and offers a tool to monitor vestibular hair cell activity in vivo . Objective To characterise features of the VM measured in vivo in guinea pigs, using a relatively simple experimental setup. Methods The VM, evoked by bone-conducted vibration (BCV), was recorded from the basal surface of either the utricular or saccular macula after surgical removal of the cochlea, in 27 guinea pigs. Results The VM remained after vestibular nerve blockade, but was abolished following end-organ destruction or death. The VM reversed polarity as the recording electrode tracked across the utricular or saccular macula surface, or through the utricular macula. The VM could be evoked by BCV stimuli of frequencies between 100 Hz and 5 kHz, and was largest to vibrations between 600 Hz and 800 Hz. Experimental manipulations demonstrated a reduction in the VM amplitude with maculae displacement, or rupture of the utricular membrane. Conclusions Results mirror those obtained in previous ex vivo studies, and further demonstrate that vestibular hair cells are sensitive to vibrations of several kilohertz. Changes in the VM with maculae displacement or rupture suggest utricular hydrops may alter vestibular hair cell sensitivity due to either mechanical or ionic changes.

The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.

Hypothermic responses accompany motion sickness in humans and can be elicited by provocative motion in rats. We aimed to determine the potential role in these responses of the efferent cholinergic vestibular innervation. To this end, we used knockout (KO) mice lacking α9 cholinoreceptor subunit predominantly expressed in the vestibular hair cells and CBA strain as a wild-type (WT) control. In WT mice, circular horizontal motion (1Hz, 4cm radius, 20min) caused rapid and dramatic falls in core body temperature and surface head temperature associated with a transient rise in the tail temperature; these responses were substantially attenuated in KO mice; changes were (WT vs. KO): for the core body temperature-5.2±0.3 vs. -2.9±0.3°C; for the head skin temperature-3.3±0.2 vs. -1.7±0.2°C; for the tail skin temperature+3.9±1.1 vs+1.1±1.2°C. There was a close correlation in the time course of cooling the body and the surface of the head. KO mice also required 25% more time to complete a balance test. We conclude: i) that the integrity of cholinergic efferent vestibular system is essential for the full expression of motion-induced hypothermia in mice, and that the role of this system is likely facilitatory; ii) that the system is involvement in control of balance, but the involvement is not major; iii) that in mice, motion-induced body cooling is mediated via increased heat flow through vasodilated tail vasculature and (likely) via reduced thermogenesis. Our results support the idea that hypothermia is a biological correlate of a nausea-like state in animals.

Vestibular sensation is essential for gaze stabilization, balance, and perception of gravity. The vestibular receptors in mammals, Type I and Type II hair cells, are located in five small organs in the inner ear. Damage to hair cells and their innervating neurons can cause crippling symptoms such as vertigo, visual field oscillation, and imbalance. In adult rodents, some Type II hair cells are regenerated and become re-innervated after damage, presenting opportunities for restoring vestibular function after hair cell damage. This article reviews features of vestibular sensory cells in mammals, including their basic properties, how they develop, and how they are replaced after damage. We discuss molecules that control vestibular hair cell regeneration and highlight areas in which our understanding of development and regeneration needs to be deepened.

Vestibular hair cells of the inner ear are specialized receptors that detect mechanical stimuli from gravity and motion via the deflection of a polarized bundle of stereocilia located on their apical cell surfaces. The orientation of stereociliary bundles is coordinated between neighboring cells by core PCP proteins including the large adhesive G-protein coupled receptor Celsr1. We show that mice lacking Celsr1 have vestibular behavioral phenotypes including circling. In addition, we show that Celsr1 is asymmetrically distributed at cell boundaries between hair cells and neighboring supporting cells in the developing vestibular and auditory sensory epithelia. In the absence of Celsr1 the stereociliary bundles of vestibular hair cells are misoriented relative to their neighbors, a phenotype that is greatest in the cristae of the semicircular canals. Since horizontal semi-circular canal defects lead to circling in other mutant mouse lines, we propose that this PCP phenotype is the cellular basis of the circling behavior in Celsr1 mutants.

In the vertebrate nervous system, ions accumulate in diffusion-limited synaptic clefts during ongoing activity. Such accumulation can be demonstrated at large appositions such as the hair cell – calyx afferent synapses present in central regions of the turtle vestibular semicircular canal epithelia. Type I hair cells influence discharge rates in their calyx afferents by modulating the potassium concentration in the synaptic cleft, [K(+)](c), which regulates potassium-sensitive conductances in both hair cell and afferent. Dual recordings from synaptic pairs have demonstrated that, despite a decreased driving force due to potassium accumulation, hair cell depolarization elicits sustained outward currents in the hair cell, and a maintained inward current in the afferent. We used kinetic and pharmacological dissection of the hair cell conductances to understand the interdependence of channel gating and permeation in the context of such restricted extracellular spaces. Hair cell depolarization leads to calcium influx and activation of a large calcium-activated potassium conductance, G(BK), that can be blocked by agents that disrupt calcium influx or buffer the elevation of [Ca(2+)](i), as well as by the specific K(Ca)1.1 blocker Iberiotoxin. Efflux of K(+) through G(BK) can rapidly elevate [K(+)](c), which speeds the activation and slows the inactivation and deactivation of a second potassium conductance, G(K(LV)). Elevation of [K(+)](c) or chelation of [Ca(2+)](c) linearizes the G(K(LV)) steady-state I–V curve, consistent with a K(+)-dependent relief of Ca(2+)-inactivation of G(K(LV)). As a result, this potassium-sensitive hair cell conductance pairs with the potassium-sensitive HCN conductance in the afferent and creates resistive coupling at the synaptic cleft.

Despite the known importance of the transcription factors ATOH1, POU4F3 and GFI1 in hair cell development and regeneration, their downstream transcriptional cascades in the inner ear remain largely unknown. Here, we have used Gfi1cre;RiboTag mice to evaluate changes to the hair cell translatome in the absence of GFI1. We identify a systematic downregulation of hair cell differentiation genes, concomitant with robust upregulation of neuronal genes in the GFI1-deficient hair cells. This includes increased expression of neuronal-associated transcription factors (e.g. Pou4f1) as well as transcription factors that serve dual roles in hair cell and neuronal development (e.g. Neurod1, Atoh1 and Insm1). We further show that the upregulated genes are consistent with the NEUROD1 regulon and are normally expressed in hair cells prior to GFI1 onset. Additionally, minimal overlap of differentially expressed genes in auditory and vestibular hair cells suggests that GFI1 serves different roles in these systems. From these data, we propose a dual mechanism for GFI1 in promoting hair cell development, consisting of repression of neuronal-associated genes as well as activation of hair cell-specific genes required for normal functional maturation.

1AbstractHair cells sense and transmit auditory, vestibular, and hydrodynamic information by converting mechanical stimuli into electrical signals. This process of mechano-electrical transduction (MET) requires a mechanically-gated channel localized in the apical stereocilia of hair cells. In mice, lipoma HMGIC fusion partner-like 5 (LHFPL5) acts as an auxiliary subunit of the MET channel whose primary role is to correctly localize PCDH15 and TMC1 to the mechanotransduction complex. Zebrafish have two lhfpl5 genes (lhfpl5a and lhfpl5b), but their individual contributions to MET channel assembly and function have not been analyzed.Here we show that the zebrafish lhfpl5 genes are expressed in discrete populations of hair cells: lhfpl5a expression is restricted to auditory and vestibular hair cells in the inner ear, while lhfpl5b expression is specific to hair cells of the lateral line organ. Consequently, lhfpl5a mutants exhibit defects in auditory and vestibular function, while disruption of lhfpl5b affects hair cells only in the lateral line neuromasts. In contrast to previous reports in mice, localization of Tmc1 does not depend upon Lhfpl5 function in either the inner ear or lateral line organ. In both lhfpl5a and lhfpl5b mutants, GFP-tagged Tmc1 and Tmc2b proteins still localize to the stereocilia of hair cells. Using a stably integrated GFP-Lhfpl5a transgene, we show that the tip link cadherins Pcdh15a and Cdh23, along with the Myo7aa motor protein, are required for correct Lhfpl5a localization at the tips of stereocilia. Our work corroborates the evolutionarily conserved co-dependence between Lhfpl5 and Pcdh15, but also reveals novel requirements for Cdh23 and Myo7aa to correctly localize Lhfpl5a. In addition, our data suggest that targeting of Tmc1 and Tmc2b proteins to stereocilia in zebrafish hair cells occurs independently of Lhfpl5 proteins.

Background:In the present study, we investigated the localization of otopetrin-2—a member of the otopetrin family that encodes proton-selective ion channels—in the human macula utricle using immunohistochemistry.Methods:Macula utricle were acquired at surgery from patients who required transmastoid labyrinthectomy for intractable vertigo due to Meniere’s disease (MD; n = 3) and/or vestibular drops attacks (VDA; n = 2) and from temporal bones (n = 2) acquired at autopsy from individuals with no balance disorders. Immunofluorescence staining with otopetrin-2 (rabbit affinity purified polyclonal antibody) and GFAP (mouse monoclonal antibody) to identify vestibular supporting cells was made in formalin fixed cryostat sections or whole microdissected utricle (for flat mount preparations). Secondary antibodies against rabbit and mouse were used for the identification of both proteins. Digital fluorescent images were obtained using a high-resolution laser confocal microscope.Results:Using cryostat sections and flat mount preparations otopetrin-2 immunofluorescence was seen as punctated signal throughout the supporting cells cytoplasm. GFAP immunofluorescence was present in the supporting cell cytoplasm. The distribution of otopetrin-2 was similar in the macula utricle obtained from MD, VDA, or autopsy normative patients.Conclusions:Otopetrin-2 was localized in supporting cells in a similar fashion that otopetrin-1 previously reported in the mouse macula utricle. The differential expression of otopetrin-2 in the supporting cells of the human macula utricle suggest an important role in the vestibular sensory periphery homeostasis and otolith maintenance.

Background Waardenburg syndrome (WS) is the consequence of an inherited autosomal dominant mutation which causes the early degeneration of intermediate cells of cochlear stria vascularis (SV) and profound hearing loss. Patients with WS may also experience primary vestibular symptoms. Most of the current WS studies did not discuss the relationship between WS and abnormal vestibular function. Our study found that a spontaneous mutant pig showed profound hearing loss and depigmentation. MITF-M, a common gene mutation causes type WS which affect the development of the intermediate cell of SV, was then identified for animal modeling. Results In this study, the degeneration of vestibular hair cells was found in pigs with MITF-M. The morphology of hair cells in vestibular organs of pigs was examined using electron microscopy from embryonic day E70 to postnatal two weeks. Significant hair cell loss in the mutant saccule was found in this study through E95 to P14. Conversely, there was no hair cell loss in either utricle or semi-circular canals. Conclusions Our study suggested that MITF-M gene mutation only affects hair cells of the saccule, but has no effect on other vestibular organs. The study also indicated that the survival of cochlear and saccular hair cells was dependent on the potassium release from the cochlear SV, but hair cells of the utricle and semi-circular canals were independent on SV.

Vestibular hair cells (V–HCs) in the inner ear have important roles and various functions. When V–HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V–HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V–HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V–HCs., Highlights • Differentiation of ES cells by vestibular cell conditioned medium (V-CM) was investigated. . • V-CM promoted expressions of vestibular hair cells (V–HCs)-related markers . • V-CM has an ability to specifically induce differentiation of ES cells into V–HCs. .

Background: Hair cells (HCs) are the mechanoreceptors for body position changes. Studies have suggested that HCs in mammals are terminally differentiated cells, which cannot regenerate after injury. Vestibular organs can generate new HCs, albeit in limited numbers. Methods: We carried out a study to analyse quantitatively and qualitatively spontaneous regeneration of vestibular HCs at different life stages. A Sox9-CreER transgenic mouse model specifically labelled with vestibular SCs was established. The proliferating cells, proliferative SCs, proliferative regenerated HCs and trans-differentiated HCs in mouse utricles of different ages were counted and analysed. Findings: We report that, from P1 to P30, the number of HCs in mouse utricles increased while the number of SCs decreased. The proliferation of SCs could be maintained until two weeks after birth. In the first week after birth, more than 30% of the total HCs were born. With an increase of age, the efficiency of HC regeneration decreased. In the adult stage, approximately 9% of new HCs still existed. Both types of HCs can be regenerated. Regenerated traced HCs are characterized by short, long, and absent bundles. Interpretation: Sox9 specifically labelled the vestibular SCs. HCs in the mouse vestibular apparatus undergo low-level turnover even in adulthood. These findings expand our understanding of sensorineural plasticity in adult vestibular organs in mammals after birth. Funding Statement: This work was supported by the National Natural Science Foundation of China (Nos. 81470687). Declaration of Interests: No authors have any professional or financial affiliations that may be regarded as a conflict of interest. Ethics Approval Statement: All animal procedures were approved by the Animal Care and Use Committee of Fudan University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Vestibular hair cells (HCs) are mechanoreceptors for the detection of head movement. Vestibular HCs of adult mammals never completely regenerate after damage, resulting in vestibular dysfunction. Overexpression of Atoh1 is effective for inducing HC regeneration. However, method of clinical feasibility and improvement of regenerative extent are both in need. Here we used an adeno-associated virus (AAV) serotype 8 vector of two different titers to overexpress Atoh1 in the injured utricles of adult mice. One month after virus inoculation, abundant myosin VIIa-positive cells and immature stereocilia were observed. Quantitative analyses revealed that Atoh1 overexpression replenished vestibular HCs in a dose-dependent manner. Vectors of a higher titer increased the number of myosin VIIa-positive cells compared to those of lower titer. Moreover, only Atoh1 overexpression in the higher titer group enhanced stereocilium regeneration, which is an important step in the maturation of regenerated HCs. Although the current treatment failed to initiate functional recovery of the animals, our results prompt further improvements in the recovery of vestibular dysfunction by AAV.

Because cyclodextrins are capable of removing cholesterol from cell membranes, there is growing interest in using these compounds to treat diseases linked to aberrant cholesterol metabolism. One compound, 2-hydroxypropyl-beta-cyclodextrin (HPβCD), is currently being evaluated as a treatment for Niemann-Pick Type C1 disease, a rare, fatal neurodegenerative disease caused by the buildup of lipids in endosomes and lysosomes. HPβCD can reduce some debilitating symptoms and extend life span, but the therapeutic doses used to treat the disease cause hearing loss. Initial studies in rodents suggested that HPβCD selectively damaged only cochlear outer hair cells during the first week post-treatment. However, our recent in vivo and in vitro studies suggested that the damage could become progressively worse and more extensive over time. To test this hypothesis, we treated rats subcutaneously with 1, 2, 3 or 4 g/kg of HPβCD and waited for 8-weeks to assess the long-term histological consequences. Our new results indicate that the two highest doses of HPβCD caused extensive damage not only to OHC, but also to inner hair cells, pillar cells and other support cells resulting in the collapse and flattening of the sensory epithelium. The 4 g/kg dose destroyed all the outer hair cells and three-fourths of the inner hair cells over the basal two-thirds of the cochlea and more than 85% of the nerve fibers in the habenula perforata and more than 80% of spiral ganglion neurons in the middle of basal turn of the cochlea. The mechanisms that lead to the delayed degeneration of inner hair cells, pillar cells, nerve fibers and spiral ganglion neurons remain poorly understood, but may be related to the loss of trophic support caused by the degeneration of sensory and/or support cells in the organ of Corti. Despite the massive damage to the cochlear sensory epithelium, the blood vessels in the stria vascularis and the vestibular hair cells in the utricle and saccule remained normal.

Little is known about the function of the cholinergic efferents innervating peripheral vestibular hair cells. We measured vestibular sensory evoked potentials (VsEPs) in α9 knockout mice, α10 knockout mice, α7 knockout mice, α9/10 and α7/9 double knockouts, and wild type controls. We also studied the morphology and ultrastructure of efferent terminals on vestibular hair cells in α9, α10 and α9/10 knockouts. Both type I and type ll vestibular hair cells express the α9 and α10 subunits. The efferent boutons on vestibular cells in α9, α10 and α9/10 knockouts appeared normal, but a quantitative analysis was not performed. Mean VsEP thresholds were significantly elevated in α9 and α9/10 knockout animals. Some α9 and α9/10 knockout animals, however, had normal or near normal thresholds, while others were greatly affected. Despite individual variability in threshold responses, latencies were consistently shortened. The double α7/9 KO resulted in decreased variance by normalizing waveforms and latencies. The phenotypes of the α7 and α10 single KOs were identical. Both α7 and α10 knockout mice evidenced normal thresholds, decreased activation latencies and had larger amplitudes compared to WT mice. The data suggest a complex interaction of nAChR receptors in regulating vestibular afferent gain and activation timing. Although the α9/10 heteromeric nAChR is an important component of vestibular efferent activity, other peripheral or central nAChRs involving the α7 subunit or α10 subunit and α9 homomeric receptors are also important. This article is protected by copyright. All rights reserved.