Hair cells in the inner ear of the pirouette and shaker 2 mutant mice

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions “cytocauds.” The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.     

Postnatal Expression of an Apamin-Sensitive K(Ca) Current in Vestibular Calyx Terminals

Afferent innervation patterns in the vestibular periphery are complex, and vestibular afferents show a large variation in their regularity of firing. Calyx fibers terminate on type I vestibular hair cells and have firing characteristics distinct from the bouton fibers that innervate type II hair cells. Whole-cell patch clamp was used to investigate ionic currents that could influence firing patterns in calyx terminals. Underlying K(Ca) conductances have been described in vestibular ganglion cells, but their presence in afferent terminals has not been investigated previously. Apamin, a selective blocker of SK-type calcium-activated K(+) channels, was tested on calyx afferent terminals isolated from gerbil semicircular canals during postnatal days 1-50. Lowering extracellular calcium or application of apamin (20-500?nM) reduced slowly activating outward currents in voltage clamp. Apamin also reduced the action potential afterhyperpolarization (AHP) in whole-cell current clamp, but only after the first two postnatal weeks. K(+) channel expression increased during the first postnatal month, and SK channels were found to contribute to the AHP, which may in turn influence discharge regularity in calyx vestibular afferents.     

Expression of α4 and β2 nicotinic acetylcholine receptor subunit mRNA and localization of α-bungarotoxin binding proteins in the rat vestibular periphery

In situ hybridization histochemistry was used to map the distribution of α2, α3, α4, and β2 nAChR subunit mRNAs throughout the peripheral vestibular system of the rat. The α4 and β2 nAChR subunit genes were co-expressed by populations of primary afferent neurons within Scarpa's ganglion, while there was no expression of the α2, α3, α4, or β2 nAChR subunit genes by type I or type II vestibular hair cells. α-bungarotoxin binding to nAChRs in the vestibular end-organs was primarily limited to the afferent chalices surrounding type I hair cells and the basal aspect of type II hair cells. These data suggest that nAChRs composed of α4 and β2 subunits are localized on afferent chalices innervating the type I vestibular hair cells and that the direct cholinergic efferent innervation of the type II vestibular hair cells utilizes nAChR composed of other subunits.     

Choline acetyltransferase immunoreactivity in the human vestibular end-organs

Acetylcholine (ACh) is believed to play a major role in the efferent vestibular system in several animal models, however no information regarding the role of ACh in the human efferent vestibular system has been published. Post-embedding immunohistochemistry in a hydrophilic resin was used to investigate the choline acetyltransferase immunoreactivity (ChATi) and acetylcholinesterase (ACHE) histochemistry in human vestibular end-organs. ChATi and AChE activity was found in numerous bouton-type terminals at the basal area of the vestibular hair cells. These terminals were found to contact type II vestibular hair cells and the afferent chalices surrounding type I hair cells. This study provides the first evidence that the human efferent vestibular axons and terminals are cholinergic.     

阳离子对豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的调制

本文旨在探讨哺乳动物前庭胆碱能传出神经系统的作用机制,应用全细胞膜片钳技术研究新鲜分离的豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的特性以及细胞内外的阳离子对ACh-敏感性电流的调制作用。结果显示,Ⅱ型前庭毛细胞对细胞外ACh敏感,ACh激活缓慢持久的外向性电流,室温下此电流的再次完全激活时间约为(60±10)s。ACh-敏感性电流的反转电位为(-66±8)mV,提示此电流主要由K+参与形成,其直接作用是使毛细胞超极化。ACh-敏感性电流对较高浓度的四乙胺(tetraethylammonium chloride,TEA)敏感,提示细胞外ACh激活钙依赖性钾电流。进一步检测细胞内外阳离子对 ACh-敏感性电流的调制作用发现,细胞外Na+和细胞内Ca2+释放不参与此电流的激活过程,而细胞外K+、细胞外Ca2+和细胞内Mg2+对ACh-敏感性电流具有重要的调制作用。进一步提示,ACh是哺乳动物前庭传出神经系统重要的神经递质。 Na+不参与ACh-敏感性电流的激活过程提示,ACh-敏感性电流可能由非α9-N型胆碱能受体(α9-nAChR)介导。ACh诱导的Ⅱ型前庭毛细胞超极化作用受细胞外Ca2+浓度和细胞内Mg2+浓度调制。     

A balance of form and function: Planar polarity and development of the vestibular maculae

The mechanosensory hair cells of the inner ear have emerged as one of the primary models for studying the development of planar polarity in vertebrates. Planar polarity is the polarized organization of cells or cellular structures in the plane of an epithelium. For hair cells, planar polarity is manifest at the subcellular level in the polarized organization of the stereociliary bundle and at the cellular level in the coordinated orientation of stereociliary bundles between adjacent cells. This latter organization is commonly called Planar Cell Polarity and has been described in the greatest detail for auditory hair cells of the cochlea. A third level of planar polarity, referred to as tissue polarity, occurs in the utricular and saccular maculae; two inner ear sensory organs that use hair cells to detect linear acceleration and gravity. In the utricle and saccule hair cells are divided between two groups that have opposite stereociliary bundle polarities and, as a result, are able to detect movements in opposite directions. Thus vestibular hair cells are a unique model system for studying planar polarity because polarization develops at three different anatomical scales in the same sensory organ. Moreover the system has the potential to be used to dissect functional interactions between molecules regulating planar polarity at each of the three levels. Here the significance of planar polarity on vestibular system function will be discussed, and the molecular mechanisms associated with development of planar polarity at each anatomical level will be reviewed. Additional aspects of planar polarity that are unique to the vestibular maculae will also be introduced.     

Electron microscope observations on in vitro cultures of the isolated fowl embryo otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Effects of KCNQ channel blockers on K(+) currents in vestibular hair cells

Linopirdine and XE991, selectiveblockers of K⁺ channels belonging to the KCNQ family, wereapplied to hair cells isolated from gerbil vestibular system and tohair cells in slices of pigeon crista. In type II hair cells, bothcompounds inhibited a slowly activating, slowly inactivating componentof the macroscopic current recruited at potentials above 60 mV. Thedissociation constants for linopirdine and XE991 block were <5µM. A similar component of the current was also blocked by 50 µMcapsaicin in gerbil type II hair cells. All three drugs blocked acurrent component that showed steady-state inactivation and abiexponential inactivation with time constants of ~300 ms and 4 s. Linopirdine (10 µM) reduced inward currents through thelow-voltage-activated K⁺ current in type I hair cells, butconcentrations up to 200 µM had little effect on steady-state outwardK⁺ current in these cells. These results suggest that KCNQchannels may be present in amniote vestibular hair cells.

     

The influence of 2.5 g exposure on the morphology of rat vestibular epithelia

Several studies have shown that altered gravity causes changes in vestibular induced reflexes and behaviour, and in vestibular morphology. How the level of gravity affects the morphology of vestibular epithelia, however, is largely unknown. Vestibular epithelia of hypergravity (HG) exposed animals and control animals were histochemically labeled for actin and tubulin (two characteristic proteins for specific cytoskeletal structures in hair cells and supporting cells). Cellular organization, cytoskeletal structure and apical cross-sectional area were investigated.     

Electron Microscope Observations on in Vitro Cultures of the Isolated Fowl Embryo Otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Coenzyme Q10 Protects Hair Cells against Aminoglycoside

It is well known that the production of free radicals is associated with sensory cell death induced by an aminoglycoside. Many researchers have reported that antioxidant reagents protect sensory cells in the inner ear, and coenzyme Q10 (CoQ10) is an antioxidant that is consumed as a health food in many countries. The purpose of this study was to investigate the role of CoQ10 in mammalian vestibular hair cell death induced by aminoglycoside. Cultured utricles of CBA/CaN mice were divided into three groups (control group, neomycin group, and neomycin + CoQ10 group). In the neomycin group, utricles were cultured with neomycin (1 mM) to induce hair cell death. In the neomycin + CoQ10 group, utricles were cultured with neomycin and water-soluble CoQ10 (30–0.3 µM). Twenty-four hours after exposure to neomycin, the cultured tissues were fixed, and vestibular hair cells were labeled using an anti-calmodulin antibody. Significantly more hair cells survived in the neomycin + CoQ10 group than in the neomycin group. These data indicate that CoQ10 protects sensory hair cells against neomycin-induced death in the mammalian vestibular epithelium; therefore, CoQ10 may be useful as a protective drug in the inner ear.     

Immunocytochemical detection of calcium-binding protein in the cochlear and vestibular hair cells of the rat

Specific antibodies raised against human cerebellar calcium-binding protein (CaBP) intensely labelled the cochlear hair cells of the rat. The vestibular hair cells also stained weakly. In both inner and outer cochlear hair cells, the cuticular plate was the most stained area. These results suggest that CaBP may prevent excessive concentrations of intracellular calcium and thus modulate some Ca2+-mediated biochemical processes, especially at the level of the cuticular plate and stereocilia; CaBP could be involved in the mechanochemical coupling of hearing or vestibular function.     

Mutations in ap1b1 Cause Mistargeting of the Na+/K+-ATPase Pump in Sensory Hair Cells

The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na⁺/K⁺-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na⁺ levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells.     

Local spiking interneurons controlling the equilibrium response in the crayfish Procambarus clarkii

1. In the crayfish brain, the responses of local spiking interneurons to body roll simulated by bending of statocyst hairs, were investigated with intracellular recording and staining techniques. The neurons had two separate branching portions in the protocerebrum and the deutocerebrum. They were named as type-I local neurons and further classified into 5 types (ac-U, vplc-U, vplc-B, vupc-U, vupc-B). 2. Vupc-U neurons showed excitatory responses and vplc-U neurons showed inhibitory responses to inward hair deflection of the statocyst ipsilateral to their deutocerebral branches. The other 3 types were of mixed populations of the interneurons showing either excitatory or inhibitory responses to the stimulation. 3. Of 10 type-I local neurons showing excitatory responses to inward hair deflection, 6 interneurons had output effects on oculomotor and/or descending neurons. All these 6 interneurons showed large EPSPs and much higher frequency of spikes to the hair stimulation than those of the other 4. All 8 type-I local neurons that showed inhibitory responses had no output effects. 4. Type-I local neurons controlled two equilibrium responses, compensatory eye movement and righting reflex, either simultaneously or independently.     

Striola magica. A functional explanation of otolith geometry

Otolith end organs of vertebrates sense linear accelerations of the head and gravitation. The hair cells on their epithelia are responsible for transduction. In mammals, the striola, parallel to the line where hair cells reverse their polarization, is a narrow region centered on a curve with curvature and torsion. It has been shown that the striolar region is functionally different from the rest, being involved in a phasic vestibular pathway. We propose a mathematical and computational model that explains the necessity of this amazing geometry for the striola to be able to carry out its function. Our hypothesis, related to the biophysics of the hair cells and to the physiology of their afferent neurons, is that striolar afferents collect information from several type I hair cells to detect the jerk in a large domain of acceleration directions. This predicts a mean number of two calyces for afferent neurons, as measured in rodents. The domain of acceleration directions sensed by our striolar model is compatible with the experimental results obtained on monkeys considering all afferents. Therefore, the main result of our study is that phasic and tonic vestibular afferents cover the same geometrical fields, but at different dynamical and frequency domains.     

GABA immunoreactivity of calyceal nerve endings in the vestibular system of the guinea pig

Summary Neurotransmitters involved in the vestibular system are largely uncharacterized. On the basis of results of earlier electrophysiological and immunohistochemical experiments, glutamate and gamma-amino-butyric acid (GABA) have been proposed in both mammalian and non-mammalian species as afferent transmitters between the sensory cell and the afferent dendrite. GABA is also suspected to act as an efferent neurotransmitter in the cochlea. We describe in this study the immunocytochemical localization of GABA within the vestibular end organs in the guinea pig. GABA immunoreactivity was found in the calyceal nerve endings surrounding type I hair cells of the vestibular epithelia. The most significant labelings were obtained in the crista ampullaris. Labeling was more difficult to observe in the utricular and saccular macula. These results contribute to the recent proposal that the calyx has a secretory function, and suggest that GABA may have a modulatory influence upon the type I hair cells.     

Glutamate Transporters EAAT4 and EAAT5 Are Expressed in Vestibular Hair Cells and Calyx Endings

Glutamate is the neurotransmitter released from hair cells. Its clearance from the synaptic cleft can shape neurotransmission and prevent excitotoxicity. This may be particularly important in the inner ear and in other sensory organs where there is a continually high rate of neurotransmitter release. In the case of most cochlear and type II vestibular hair cells, clearance involves the diffusion of glutamate to supporting cells, where it is taken up by EAAT1 (GLAST), a glutamate transporter. A similar mechanism cannot work in vestibular type I hair cells as the presence of calyx endings separates supporting cells from hair-cell synapses. Because of this arrangement, it has been conjectured that a glutamate transporter must be present in the type I hair cell, the calyx ending, or both. Using whole-cell patch-clamp recordings, we demonstrate that a glutamate-activated anion current, attributable to a high-affinity glutamate transporter and blocked by DL-TBOA, is expressed in type I, but not in type II hair cells. Molecular investigations reveal that EAAT4 and EAAT5, two glutamate transporters that could underlie the anion current, are expressed in both type I and type II hair cells and in calyx endings. EAAT4 has been thought to be expressed almost exclusively in the cerebellum and EAAT5 in the retina. Our results show that these two transporters have a wider distribution in mice. This is the first demonstration of the presence of transporters in hair cells and provides one of the few examples of EAATs in presynaptic elements.     

Mice with altered KCNQ4 K+ channels implicate sensory outer hair cells in human progressive deafness

KCNQ4 is an M-type K+ channel expressed in sensory hair cells of the inner ear and in the central auditory pathway. KCNQ4 mutations underlie human DFNA2 dominant progressive hearing loss. We now generated mice in which the KCNQ4 gene was disrupted or carried a dominant negative DFNA2 mutation. Although KCNQ4 is strongly expressed in vestibular hair cells, vestibular function appeared normal. Auditory function was only slightly impaired initially. It then declined over several weeks in Kcnq4-/- mice and over several months in mice carrying the dominant negative allele. This progressive hearing loss was paralleled by a selective degeneration of outer hair cells (OHCs). KCNQ4 disruption abolished the I(K,n) current of OHCs. The ensuing depolarization of OHCs impaired sound amplification. Inner hair cells and their afferent synapses remained mostly intact. These cells were only slightly depolarized and showed near-normal presynaptic function. We conclude that the hearing loss in DFNA2 is predominantly caused by a slow degeneration of OHCs resulting from chronic depolarization.