LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel.

During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5-/- knockout mice with those in Lhfpl5+/- heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/- was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5-/- mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5-/-, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5-/- doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

Optogenetics Reveals Roles for Supporting Cells in Force Transmission to and From Outer Hair Cells in the Mouse Cochlea.

Outer hair cells (OHCs) of the organ of Corti (OoC), acting as bidirectional cellular mechanoelectrical transducers, generate, receive, and exchange forces with other major elements of the cochlear partition, including the sensory inner hair cells (IHCs). Force exchange is mediated via a supporting cell scaffold, including Deiters' (DC) and outer pillar cells (OPC), to enable the sensitivity and exquisite frequency selectivity of the mammalian cochlea and to transmit its responses to the auditory nerve. To selectively activate DCs and OPCs in male and female mice, we conditionally expressed in them a hyperpolarizing halorhodopsin (HOP), a light-gated inward chloride ion pump, and measured extracellular receptor potentials (ERPs) and their DC component (ERPDCs) from the cortilymph, which fills the OoC fluid spaces, and compared the responses with similar potentials from HOP-/- littermates. The compound action potentials (CAP) of the auditory nerve were measured as an indication of IHC activity and transmission of cochlear responses to the CNS. HOP light-activated hyperpolarization of DCs and OPCs suppressed cochlear amplification through changing the timing of its feedback, altered basilar membrane (BM) responses to tones at all measured levels and frequencies, and reduced IHC excitation. HOP activation findings reported here complement recent studies that revealed channelrhodopsin activation depolarized DCs and OPCs and effectively bypassed, rather than blocked, the control of OHC mechanical and electrical responses to sound and their contribution to timed and directed electromechanical feedback to the mammalian cochlea. Moreover, our findings identify DCs and OPCs as potential targets for the treatment of noise-induced hearing loss.

In situ regeneration of inner hair cells in the damaged cochlea by temporally regulated co-expression of Atoh1 and Tbx2.

Cochlear inner hair cells (IHCs) are primary sound receptors, and are therefore a target for developing treatments for hearing impairment. IHC regeneration in vivo has been widely attempted, although not yet in the IHC-damaged cochlea. Moreover, the extent to which new IHCs resemble wild-type IHCs remains unclear, as is the ability of new IHCs to improve hearing. Here, we have developed an in vivo mouse model wherein wild-type IHCs were pre-damaged and nonsensory supporting cells were transformed into IHCs by ectopically expressing Atoh1 transiently and Tbx2 permanently. Notably, the new IHCs expressed the functional marker vGlut3 and presented similar transcriptomic and electrophysiological properties to wild-type IHCs. Furthermore, the formation efficiency and maturity of new IHCs were higher than those previously reported, although marked hearing improvement was not achieved, at least partly due to defective mechanoelectrical transduction (MET) in new IHCs. Thus, we have successfully regenerated new IHCs resembling wild-type IHCs in many respects in the damaged cochlea. Our findings suggest that the defective MET is a critical barrier that prevents the restoration of hearing capacity and should thus facilitate future IHC regeneration studies.

Multisensory gaze stabilization in response to subchronic alteration of vestibular type I hair cells.

The functional complementarity of the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) allows for optimal combined gaze stabilization responses (CGR) in light. While sensory substitution has been reported following complete vestibular loss, the capacity of the central vestibular system to compensate for partial peripheral vestibular loss remains to be determined. Here, we first demonstrate the efficacy of a 6-week subchronic ototoxic protocol in inducing transient and partial vestibular loss which equally affects the canal- and otolith-dependent VORs. Immunostaining of hair cells in the vestibular sensory epithelia revealed that organ-specific alteration of type I, but not type II, hair cells correlates with functional impairments. The decrease in VOR performance is paralleled with an increase in the gain of the OKR occurring in a specific range of frequencies where VOR normally dominates gaze stabilization, compatible with a sensory substitution process. Comparison of unimodal OKR or VOR versus bimodal CGR revealed that visuo-vestibular interactions remain reduced despite a significant recovery in the VOR. Modeling and sweep-based analysis revealed that the differential capacity to optimally combine OKR and VOR correlates with the reproducibility of the VOR responses. Overall, these results shed light on the multisensory reweighting occurring in pathologies with fluctuating peripheral vestibular malfunction.

Ca2+ regulation of glutamate release from inner hair cells of hearing mice.

In our hearing organ, sound is encoded at ribbon synapses formed by inner hair cells (IHCs) and spiral ganglion neurons (SGNs). How the underlying synaptic vesicle (SV) release is controlled by Ca2+ in IHCs of hearing animals remained to be investigated. Here, we performed patch-clamp SGN recordings of the initial rate of release evoked by brief IHC Ca2+-influx in an ex vivo cochlear preparation from hearing mice. We aimed to closely mimic physiological conditions by perforated-patch recordings from IHCs kept at the physiological resting potential and at body temperature. We found release to relate supralinearly to Ca2+-influx (power, m: 4.3) when manipulating the [Ca2+] available for SV release by Zn2+-flicker-blocking of the single Ca2+-channel current. In contrast, a near linear Ca2+ dependence (m: 1.2 to 1.5) was observed when varying the number of open Ca2+-channels during deactivating Ca2+-currents and by dihydropyridine channel-inhibition. Concurrent changes of number and current of open Ca2+-channels over the range of physiological depolarizations revealed m: 1.8. These findings indicate that SV release requires ~4 Ca2+-ions to bind to their Ca2+-sensor of fusion. We interpret the near linear Ca2+-dependence of release during manipulations that change the number of open Ca2+-channels to reflect control of SV release by the high [Ca2+] in the Ca2+-nanodomain of one or few nearby Ca2+-channels. We propose that a combination of Ca2+ nanodomain control and supralinear intrinsic Ca2+-dependence of fusion optimally links SV release to the timing and amplitude of the IHC receptor potential and separates it from other IHC Ca2+-signals unrelated to afferent synaptic transmission.

Evidence That Ultrafast Nonquantal Transmission Underlies Synchronized Vestibular Action Potential Generation.

Amniotes evolved a unique postsynaptic terminal in the inner ear vestibular organs called the calyx that receives both quantal and nonquantal (NQ) synaptic inputs from Type I sensory hair cells. The nonquantal synaptic current includes an ultrafast component that has been hypothesized to underlie the exceptionally high synchronization index (vector strength) of vestibular afferent neurons in response to sound and vibration. Here, we present three lines of evidence supporting the hypothesis that nonquantal transmission is responsible for synchronized vestibular action potentials of short latency in the guinea pig utricle of either sex. First, synchronized vestibular nerve responses are unchanged after administration of the AMPA receptor antagonist CNQX, while auditory nerve responses are completely abolished. Second, stimulus evoked vestibular nerve compound action potentials (vCAP) are shown to occur without measurable synaptic delay and three times shorter than the latency of auditory nerve compound action potentials (cCAP), relative to the generation of extracellular receptor potentials. Third, paired-pulse stimuli designed to deplete the readily releasable pool (RRP) of synaptic vesicles in hair cells reveal forward masking in guinea pig auditory cCAPs, but a complete lack of forward masking in vestibular vCAPs. Results support the conclusion that the fast component of nonquantal transmission at calyceal synapses is indefatigable and responsible for ultrafast responses of vestibular organs evoked by transient stimuli.SIGNIFICANCE STATEMENT The mammalian vestibular system drives some of the fastest reflex pathways in the nervous system, ensuring stable gaze and postural control for locomotion on land. To achieve this, terrestrial amniotes evolved a large, unique calyx afferent terminal which completely envelopes one or more presynaptic vestibular hair cells, which transmits mechanosensory signals mediated by quantal and nonquantal (NQ) synaptic transmission. We present several lines of evidence in the guinea pig which reveals the most sensitive vestibular afferents are remarkably fast, much faster than their auditory nerve counterparts. Here, we present neurophysiological and pharmacological evidence that demonstrates this vestibular speed advantage arises from ultrafast NQ electrical synaptic transmission from Type I hair cells to their calyx partners.

Molecular signatures define subtypes of auditory afferents with distinct peripheral projection patterns and physiological properties.

Type I spiral ganglion neurons (SGNs) are the auditory afferents that transmit sound information from cochlear inner hair cells (IHCs) to the brainstem. These afferents consist of physiological subtypes that differ in their spontaneous firing rate (SR), activation threshold, and dynamic range and have been described as low, medium, and high SR fibers. Lately, single-cell RNA sequencing experiments have revealed three molecularly defined type I SGN subtypes. The extent to which physiological type I SGN subtypes correspond to molecularly defined subtypes is unclear. To address this question, we have generated mouse lines expressing CreERT2 in SGN subtypes that allow for a physiological assessment of molecular subtypes. We show that Lypd1-CreERT2 expressing SGNs represent a well-defined group of neurons that preferentially innervate the IHC modiolar side and exhibit a narrow range of low SRs. In contrast, Calb2-CreERT2 expressing SGNs preferentially innervate the IHC pillar side and exhibit a wider range of SRs, thus suggesting that a strict stratification of all SGNs into three molecular subclasses is not obvious, at least not with the CreERT2 tools used here. Genetically marked neuronal subtypes refine their innervation specificity onto IHCs postnatally during the time when activity is required to refine their molecular phenotype. Type I SGNs thus consist of genetically defined subtypes with distinct physiological properties and innervation patterns. The molecular subtype-specific lines characterized here will provide important tools for investigating the role of the physiologically distinct type I SGNs in encoding sound signals.

Development and evaluation of helper dependent adenoviral vectors for inner ear gene delivery.

Viral vector gene therapy is an attractive strategy to treat hearing loss. Since hearing loss is due to a variety of pathogenic signaling cascades in distinct cells, viral vectors that can express large or multiple genes in a cell-type specific manner are needed. Helper-dependent adenoviral vectors (HdAd) are safe viral vectors with a large packaging capacity (-36 kb). Despite the potential of HdAd, its use in the inner ear is largely unexplored. Therefore, to evaluate the utility of HdAd for inner ear gene therapy, we created two HdAd vectors that use distinct cellular receptors for transduction: HdAd Serotype Type 5 (HdAd5), the Coxsackie-Adenovirus Receptor (CAR) and a chimeric HdAd 5/35, the human CD46+ receptor (hCD46). We delivered these vectors through the round window (RW) or scala media in CBA/J, C57Bl6/J and hCD46 transgenic mice. Immunostaining in conjunction with confocal microscopy of cochlear sections revealed that multiple cell types were transduced using HdAd5 and HdAd 5/35 in all mouse models. Delivery of HdAd5 via RW in the C57Bl/6 J or CBA/J cochlea resulted in transduced mesenchymal cells of the peri­lymphatic lining and modiolar region while scala media delivery resulted in transduction of supporting cells and inner hair cells. Hd5/35 transduction was CD46 dependent and RW delivery of HdAd5/35 in the hCD46 mouse model resulted in a similar transduction pattern as HdAd5 in the peri­lymphatic lining and modiolar region in the cochlea. Our data indicate that HdAd vectors are promising vectors for use in inner ear gene therapy to treat some causes of hearing loss.

Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution.

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.

Expression of hyperpolarization-activated current (Ih) in zonally defined vestibular calyx terminals of the crista.

Calyx terminals make afferent synapses with type I hair cells in vestibular epithelia and express diverse ionic conductances that influence action potential generation and discharge regularity in vestibular afferent neurons. Here we investigated the expression of hyperpolarization-activated current (Ih) in calyx terminals in central and peripheral zones of mature gerbil crista slices, using whole cell patch-clamp recordings. Slowly activating Ih was present in >80% calyces tested in both zones. Peak Ih and half-activation voltages were not significantly different; however, Ih activated with a faster time course in peripheral compared with central zone calyces. Calyx Ih in both zones was blocked by 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD7288; 100 µM), and the resting membrane potential became more hyperpolarized. In the presence of dibutyryl-cAMP (dB-cAMP), peak Ih was increased, activation kinetics became faster, and the voltage of half-activation was more depolarized compared with control calyces. In current clamp, calyces from both zones showed three different categories of firing: spontaneous firing, phasic firing where a single action potential was evoked after a hyperpolarizing pulse, or a single evoked action potential followed by membrane potential oscillations. In the absence of Ih, the latency to peak of the action potential increased; Ih produces a small depolarizing current that facilitates firing by driving the membrane potential closer to threshold. Immunostaining showed the expression of HCN2 subunits in calyx terminals. We conclude that Ih is found in calyx terminals across the crista and could influence conventional and novel forms of synaptic transmission at the type I hair cell-calyx synapse.NEW & NOTEWORTHY Calyx afferent terminals make synapses with vestibular hair cells and express diverse conductances that impact action potential firing in vestibular primary afferents. Conventional and nonconventional synaptic transmission modes are influenced by hyperpolarization-activated current (Ih), but regional differences were previously unexplored. We show that Ih is present in both central and peripheral calyces of the mammalian crista. Ih produces a small depolarizing resting current that facilitates firing by driving the membrane potential closer to threshold.

The vestibular calyceal junction is dismantled following subchronic streptomycin in rats and sensory epithelium stress in humans.

Hair cell (HC) loss by epithelial extrusion has been described to occur in the rodent vestibular system during chronic 3,3'-iminodipropionitrile (IDPN) ototoxicity. This is preceded by dismantlement of the calyceal junction in the contact between type I HC (HCI) and calyx afferent terminals. Here, we evaluated whether these phenomena have wider significance. First, we studied rats receiving seven different doses of streptomycin, ranging from 100 to 800 mg/kg/day, for 3-8 weeks. Streptomycin caused loss of vestibular function associated with partial loss of HCI and decreased expression of contactin-associated protein (CASPR1), denoting calyceal junction dismantlement, in the calyces encasing the surviving HCI. Additional molecular and ultrastructural data supported the conclusion that HC-calyx detachment precede HCI loss by extrusion. Animals allowed to survive after the treatment showed functional recuperation and rebuilding of the calyceal junction. Second, we evaluated human sensory epithelia obtained during therapeutic labyrinthectomies and trans-labyrinthine tumour excisions. Some samples showed abnormal CASPR1 label strongly suggestive of calyceal junction dismantlement. Therefore, reversible dismantlement of the vestibular calyceal junction may be a common response triggered by chronic stress, including ototoxic stress, before HCI loss. This may partly explain clinical observations of reversion in function loss after aminoglycoside exposure.

CIB2 and CIB3 Regulate Stereocilia Maintenance and Mechanoelectrical Transduction in Mouse Vestibular Hair Cells.

The mechanoelectrical transduction (MET) protein complex in the inner-ear hair cells is essential for hearing and balance perception. Calcium and integrin-binding protein 2 (CIB2) has been reported to be a component of MET complex, and loss of CIB2 completely abolishes MET currents in auditory hair cells, causing profound congenital hearing loss. However, loss of CIB2 does not affect MET currents in vestibular hair cells (VHCs) as well as general balance function. Here, we show that CIB2 and CIB3 act redundantly to regulate MET in VHCs, as MET currents are completely abolished in the VHCs of Cib2/Cib3 double knock-out mice of either sex. Furthermore, we show that Cib2 and Cib3 transcripts have complementary expression patterns in the vestibular maculae, and that they play different roles in stereocilia maintenance in VHCs. Cib2 transcripts are highly expressed in the striolar region, and knock-out of Cib2 affects stereocilia maintenance in striolar VHCs. In contrast, Cib3 transcripts are highly expressed in the extrastriolar region, and knock-out of Cib3 mainly affects stereocilia maintenance in extrastriolar VHCs. Simultaneous knock-out of Cib2 and Cib3 affects stereocilia maintenance in all VHCs and leads to severe balance deficits. Taken together, our present work reveals that CIB2 and CIB3 are important for stereocilia maintenance as well as MET in mouse VHCs.SIGNIFICANCE STATEMENT Calcium and integrin-binding protein 2 (CIB2) is an important component of mechanoelectrical transduction (MET) complex, and loss of CIB2 completely abolishes MET in auditory hair cells. However, MET is unaffected in Cib2 knock-out vestibular hair cells (VHCs). In the present work, we show that CIB3 could compensate for the loss of CIB2 in VHCs, and Cib2/Cib3 double knock-out completely abolishes MET in VHCs. Interestingly, CIB2 and CIB3 could also regulate VHC stereocilia maintenance in a nonredundant way. Cib2 and Cib3 transcripts are highly expressed in the striolar and extrastriolar regions, respectively. Stereocilia maintenance and balance function are differently affected in Cib2 or Cib3 knock-out mice. In conclusion, our data suggest that CIB2 and CIB3 are important for stereocilia maintenance and MET in mouse VHCs.

The hair cell analysis toolbox is a precise and fully automated pipeline for whole cochlea hair cell quantification.

Our sense of hearing is mediated by sensory hair cells, precisely arranged and highly specialized cells subdivided into outer hair cells (OHCs) and inner hair cells (IHCs). Light microscopy tools allow for imaging of auditory hair cells along the full length of the cochlea, often yielding more data than feasible to manually analyze. Currently, there are no widely applicable tools for fast, unsupervised, unbiased, and comprehensive image analysis of auditory hair cells that work well either with imaging datasets containing an entire cochlea or smaller sampled regions. Here, we present a highly accurate machine learning-based hair cell analysis toolbox (HCAT) for the comprehensive analysis of whole cochleae (or smaller regions of interest) across light microscopy imaging modalities and species. The HCAT is a software that automates common image analysis tasks such as counting hair cells, classifying them by subtype (IHCs versus OHCs), determining their best frequency based on their location along the cochlea, and generating cochleograms. These automated tools remove a considerable barrier in cochlear image analysis, allowing for faster, unbiased, and more comprehensive data analysis practices. Furthermore, HCAT can serve as a template for deep learning-based detection tasks in other types of biological tissue: With some training data, HCAT's core codebase can be trained to develop a custom deep learning detection model for any object on an image.

The expression of p27 in the adult vestibular sensory organs and its possible roles.

Vestibular hair cells (HCs) located in the inner ear are the receptors of vestibular sensory, which facilitates the human sense of balance. The detailed differentiation pattern and maturation process of the vestibular HCs are unclear now. p27, a cyclin/CDK inhibitor, plays a critical role in regulating the exit of cell cycle. We found that p27 was continuously expressed in the terminally differentiated and mature vestibular HCs using p27-P2A-iCreER/+; Rosa26-LSL-tdTomato/+ mice, suggesting p27 might have novel roles independent of its CDK inhibitory action. p27 is also reported to be associated with cell differentiation, cell migration and cell survival. We further explored the difference of p27 expression between two subtypes of vestibular HCs, and found that the proportion of p27-tdTomato positive type I vestibular HCs increased gradually along the subtype determination and maturation of vestibular HCs, suggesting that p27 might play a role in the HC subtype differentiation, maturation and function acquirement.

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

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

Molecular and Functional Changes to Postsynaptic Cholinergic Signaling in the Vestibular Sensory Organs of Aging C57BL/6 Mice.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Nonquantal transmission at the vestibular hair cell-calyx synapse: KLV currents modulate fast electrical and slow K+ potentials.

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 presynaptic hair cell to the postsynaptic 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 experimental observations of NQT and identifies features that support 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.

Fbxo2CreERT2: A new model for targeting cells in the neonatal and mature inner ear.

The mammalian inner ear contains six sensory patches that allow detection of auditory stimuli as well as movement and balance. Much research has focused on the organ of Corti, the sensory organ of the cochlea that detects sound. Unfortunately, these cells are difficult to access in vivo, especially in the mature animal, but the development of genetically modified mouse models, including Cre/Lox mice, has improved the ability to label, purify or manipulate these cells. Here, we describe a new tamoxifen-inducible CreER mouse line, the Fbxo2CreERT2 mouse, that can be used to specifically manipulate cells throughout the cochlear duct of the neonatal and mature cochlear epithelium. In vestibular sensory epithelia, Fbxo2CreERT2-mediated recombination occurs in many hair cells and more rarely in supporting cells of neonatal and adult mice, with a higher rate of Fbxo2CreERT2 induction in type 1 versus type 2 hair cells in adults. Fbxo2CreERT2 mice, therefore, are a new tool for the specific manipulation of epithelial cells of the inner ear and targeted manipulation of vestibular type 1 hair cells.

Culture of organoids with vestibular cell-derived factors promotes differentiation of embryonic stem cells into inner ear vestibular hair cells.

Vestibular hair cells (V-HCs) residing in the inner ear have important roles related to balance. Although differentiation of pluripotent stem cells into HCs has been shown, an effective method has yet to be established. We previously reported that use of vestibular cell-derived conditioned medium (V-CM) was helpful to induce embryonic stem (ES) cells to differentiate into V-HC-like cells in two-dimensional (2D) cultures of ES-derived embryoid bodies (EBs). In the present report, V-CM was used with three-dimensional (3D) cultures of EBs, which resulted in augmented expression of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5), but not of the cochlear HC-related marker Lmod3. Gene expression analyses of both 2D and 3D EBs cultured for two weeks revealed a greater level of augmented induction of HC-related markers in the 3D-cultured EBs. These results indicate that a 3D culture in combination with use of V-CM is an effective method for producing V-HCs.

Sox2 is required in supporting cells for normal levels of vestibular hair cell regeneration in adult mice.

Sox2 is a transcription factor that is necessary in the mammalian inner ear for development of sensory hair cells and supporting cells. Sox2 is expressed in supporting cells of adult mammals, but its function in this context is poorly understood. Given its role in the developing inner ear, we hypothesized that Sox2 is required in vestibular supporting cells for regeneration of type II hair cells after damage. Using adult mice, we deleted Sox2 from Sox9-CreER-expressing supporting cells prior to diphtheria toxin-mediated hair cell destruction and used fate-mapping to assess regeneration. In utricles of control mice with normal Sox2 expression, supporting cells regenerated nearly 200 hair cells by 3 weeks post-damage, which doubled by 12 weeks. In contrast, mice with Sox2 deletion from supporting cells had approximately 20 fate-mapped hair cells at 3 weeks post-damage, and this number did not change significantly by 12 weeks, indicating regeneration was dramatically curtailed. We made similar observations for saccules and ampullae. We found no evidence that supporting cells lacking Sox2 had altered cellular density, morphology, or ultrastructure. However, some Sox2-negative supporting cell nuclei appeared to migrate apically but did not turn on hair cell markers, and type I hair cell survival was higher. Sox2 heterozygotes also had reduced regeneration in utricles, but more hair cells were replaced than mice with Sox2 deletion. Our study determined that Sox2 is required in supporting cells for normal levels of vestibular hair cell regeneration but found no other major requirements for Sox2 in adult supporting cells.