Three-dimensional Architecture of Hair-bundle Linkages Revealed by Electron-microscopic Tomography

The senses of hearing and balance rest upon mechanoelectrical transduction by the hair bundles of hair cells in the inner ear. Located at the apical cellular surface, each hair bundle comprises several tens of stereocilia and a single kinocilium that are interconnected by extracellular proteinaceous links. Using electron-microscopic tomography of bullfrog saccular sensory epithelia, we examined the three-dimensional structures of basal links, kinociliary links, and tip links. We observed significant differences in the appearances and dimensions of these three structures and found two distinct populations of tip links suggestive of the involvement of different proteins, splice variants, or protein-protein interactions. We noted auxiliary links connecting the upper portions of tip links to the taller stereocilia. Tip links and auxiliary links show a tendency to adopt a globular conformation when disconnected from the membrane surface.     

雏鸡基底乳头Fas蛋白表达与细胞凋亡的关系

目的 :研究耳毒性药物致雏鸡内耳损伤过程中细胞凋亡与Fas蛋白表达的关系。方法 :应用卡那霉素 10 0mg/ (kg·d)皮下注射 ,连续 10d ,建立雏鸡内耳损伤模型。在雏鸡内耳损伤后 1、3、7、14、2 1d采用免疫组化方法、TUNEL法 ,分别对基底乳头Fas蛋白免疫反应阳性细胞和凋亡细胞分布进行观察。结果 :TUNEL阳性细胞主要分布在雏鸡基底乳头腔面毛细胞层 ,较高水平标记发生在停药后第 7天 ,停药后第 14天 ,TUNEL阳性细胞开始减少。Fas蛋白表达从停药后第 1天开始增高 ,停药后第 3天表达水平达高峰 ,随后逐渐下降。结论 :Fas蛋白在雏鸡基底乳头损伤后的高水平表达与毛细胞凋亡紧密相关 ,可能参与了损伤过程中的毛细胞凋亡调控。     

豚鼠Ⅱ型前庭毛细胞乙酰胆碱敏感性钾电流的钙离子依赖性

目的研究豚鼠Ⅱ型前庭毛细胞乙酰胆碱(acetylcholine,ACh)敏感性钾电流对钙离子的依赖性。方法应用全细胞膜片钳技术研究新鲜单离的豚鼠Ⅱ型前庭毛细胞ACh-敏感性钾电流在细胞内外钙离子浓度改变时其电流幅值的变化。结果①细胞外ACh激活一缓慢持久的外向性钾电流,其反转电位为(-70±10)mV;②ACh-敏感性钾电流电流对细胞外四乙胺(10mmol/L)敏感,而对细胞外4-氨基吡啶(100μmol/L)不敏感;③ACh-敏感性钾电流的幅值大小依赖于细胞外的钙离子浓度,无钙外液中ACh激活一很小的电流,4mmol/L外钙溶液中ACh-敏感性钾电流的幅值达到最大值,而0·5mmol/L外钙溶液中ACh-敏感性钾电流的幅值抑制至(36·5±6·5)%;④细胞内三磷酸肌醇-钙离子释放过程不参与ACh-敏感性钾电流的激活,细胞内透析肝素8mg/ml后30min,ACh-敏感性钾电流的幅值没有明显改变;⑤ACh敏感性钾电流对钙通道阻断剂Cd2+敏感。结论豚鼠Ⅱ型前庭毛细胞ACh-敏感性钾电流的激活依赖于细胞外的钙离子浓度,ACh与豚鼠Ⅱ型前庭毛细胞胆碱能受体结合后,首先激活膜上钙通道引起细胞外钙离子内流,毛细胞内游离钙离子浓度的升高进一步激活钙依赖性钾电流。     

斯里兰卡肉桂碱受体在不同鼠龄大鼠耳蜗中的表达差异

目的 研究不同鼠龄大鼠耳蜗内斯里兰卡肉桂碱受体(ryanodine receptor,RyR)的差异表达.探讨RyR表达与耳蜗发育成熟的关系.方法 分别选取出生后1天(P1)、5天(P5)、10天(P10)、14天(P14)、28天(P28)以及成年SD大鼠(＞2月龄)各5只耳蜗作冰冻切片.运用免疫荧光的方法 ,比较各组大鼠耳蜗组织RyR的表达差异.结果 P1组和P5组大鼠耳蜗Corti器内RyR表达不明显,P10组出现的RyR染色均匀,而P14组大鼠耳蜗Corti器内RyR的表达出现明显特征性变化,毛细胞突触区RyR强表达,而支持细胞内的表达相对较为广泛.大鼠出生后耳蜗螺旋神经元内RyR的表达由广泛的胞内表达,逐渐趋近细胞膜突触区.结论 RyR在大鼠出生后14天表达基本成熟,与耳蜗的发育成熟基本保持一致.感觉毛细胞和螺旋神经元的RyR表达可能与神经传递等功能相关.在发育早期的螺旋神经元细胞中RyR的广泛表达,可能参与了螺旋神经元发育成熟中的细胞凋亡过程.     

Plasma membrane Ca2+-ATPase isoforms in frog crista ampullaris: identification of PMCA1 and PMCA2 specific splice variants

Ca2+ ions play a pivotal role in inner ear hair cells as they are involved from the mechano-electrical transduction to the transmitter release. Most of the Ca2+ that enters into hair cells via mechano-transduction and voltage-gated channels is extruded by the plasma membrane Ca2+-ATPases (PMCAs) that operate in both apical and basal cellular compartments. Here, we determined the identity and distribution of PMCA isoforms in frog crista ampullaris: we showed that PMCA1, PMCA2 and PMCA3 are expressed, while PMCA4 appears to be negligible. We also identify PMCA1bx, PMCA2av and PMCA2bv as the major splice variants produced from PMCA1 and PMCA2 genes. PMCA2av appears to be the major Ca2+-pump operating at the apical pole of the cell, even if PMCA1b is also expressed in the stereocilia. PMCA1bx is, instead, the principal PMCA of hair cell basolateral compartment, where it is expressed together with PMCA2 (probably PMCA2bv) and PMCA3. Frog crista ampullaris hair cells lack a Na/Ca exchanger, therefore PMCAs are the only mechanism of Ca2+ extrusion. The coexpression of specific isozymes in the different cellular compartments responds to the need of a fine regulation of both basal and dynamic Ca2+ levels at the apical and basal pole of the cell.     

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.     

Uptake of Gentamicin by Bullfrog Saccular Hair Cells <Emphasis Type="Italic">in vitro</Emphasis>

Vertebrate sensory hair cells in the inner ear are pharmacologically sensitive to aminoglycoside antibiotics. Although the ototoxicity of aminoglycosides is well known, the route of drug uptake by hair cells and mechanisms of cytotoxicity remain poorly understood. Previously published studies have documented the intracellular distribution of gentamicin using immunocytochemical, electron microscopic, and autoradiographic methods. In this article, we compare the subcellular distribution of fluorescently conjugated gentamicin (gentamicin–Texas Red, GTTR) with immunolabeled gentamicin using confocal or electron microscopy. Gentamicin (detected by postfixation immunocytochemistry) and GTTR were rapidly taken up by hair cells throughout the bullfrog saccular explant in vitro and preferentially in peripheral hair cells. Immunolabeled gentamicin and GTTR were observed at the apical membranes of hair cells, particularly in their hair bundles. GTTR was also identified within a variety of subcellular compartments within hair cells, including lysosomes, mitochondria, Golgi bodies, endoplasmic reticulum, and nuclei, and in similar structures by immunoelectron microscopy. The distributions of GTTR and immunolabeled gentamicin are largely identical and corroborate a variety of published immunocytochemical and autoradiography studies. Thus, GTTR is a valid fluorescent probe with which to investigate the pharmacokinetics and mechanisms of gentamicin accumulation.     

Biochemical mechanisms affecting susceptibility to noise-induced hearing loss

In magnesium (Mg)-deficient rats and guinea pigs, noise-induced hearing loss (NIHL) was found to be correlated to the decrease of Mg in serum and perilymph. Also, in noise-exposed humans, NIHL increased with decreasing serum Mg. During the process of mechanoelectrical transduction within the hair cells in the inner ear, membrane permeability of K+ and Ca2+ will transiently increase. Mg deficiency may additionally increase membrane permeability and, therefore, energy-dependent K+ and Ca2+ turnover. The increased release of catecholamines in Mg deficiency may affect the hair cells, either directly by increasing the intracellular concentration of free Ca2+ and/or indirectly by reducing the blood flow. Also, thromboxane A2, which is increased in Mg deficiency, may reduce the blood flow in the inner ear. By these mechanisms, Mg deficiency may cause energy depletion and irreversible damage to the hair cells.     

Ionic changes in cochlear endolymph of the guinea pig induced by acoustic injury

The effects of acoustic overstimulation on the endocochlear potential (EP) and on concentrations of ions (K+, Na+, Cl-, H+, HCO3-, and Ca2+) in endolymph were investigated using ion-selective microelectrodes. A slight but significant elevation of the EP and alkalinization of the endolymph were induced by acoustic overstimulation, whereas there was little change in the K+, Na+, Cl-, and HCO3- concentrations. The changes in H+ and HCO3- concentrations implied a depression of PCO2, suggesting an increase in blood flow to the cochlea. On the other hand, the Ca2+ concentration increased abruptly to 48 times the pre-exposure value. In contrast, no significant change in the Ca2+ concentration was observed in cochleae with damaged hair cells. We discuss the mechanism of the tone-induced Ca2+ elevation in endolymph and its effect on hearing acuity.     

温度变化对豚鼠离体前庭毛细胞内钙离子浓度的影响

目的 为探讨温度改变诱发前庭眼震的机理。方法 酶孵育后机械分离成功豚鼠前庭毛细胞（ＶＨＣ）３４个，Ｆｌｕｏ－３荧光染色后，改变其外环境的温度，用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下及温度改变时ＶＨＣ内钙离子浓度（［Ｃａ＾２＋］ｉ）的变化。结果 将分离的ＶＨＣ分为两型，ＬＳＣＭ下钙离子分布图像与相差显微镜下所见细胞外形相似，其钙离子分布在核区略高。１８／２２的Ｉ型与７／１２的Ⅱ型ＶＨＣ其细     

An in vitro rapid evaluation of drug-induced ototoxicity and of reductive effect of calcium on aminoglycoside ototoxicity using organ culture

The ototoxicity of aminoglycoside and anticancer platinum drug was analysed using an organ culture system. The effect of calcium antagonism on aminoglycoside ototoxicity was investigated by the same system. The inner ears of mice, 16-day embryos, were cultured for 5 days with or without gentamicin (GM) or kanamycin (KM) or streptomycin (SM), or ribostamycin (RSM), including 1, 10, 100, or 1000 micrograms/ml respectively. The 21st gestational day-inner ear was cultured in vitro during 4 days with or without Cisplatin (CDDP) or platinum analog DWA2114R (DWA), including 0.1, 1, or 10 micrograms/ml, respectively. The 16th gestational day-inner ear was cultured in vitro for 5 days with 10 micrograms/ml KM, adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium. The damages of crista ampullaris and macula utriculi of cultured inner ear were estimated according to the ototoxicity score based on morphological changes by a light microscopic observation of serial sections of the materials. We defined four grades for the damages according to the following criteria; grade 1: damage of apical surface of the hair cells, grade 2: the existence of debris in the endolymph space, grade 3: disappearance of the hair cells, grade 4: degeneration of the supporting cells. Using this system following results were obtained: 1) the effect of aminoglycoside was dose dependent, 2) the order of ototoxicity was following; GM greater than KM greater than SM greater than RSM, 3) the drug concentration of 1000 micrograms/ml is sufficient to study its ototoxic potential in this system, 4) the effect of both CDDP and DWA was obvious at a concentration of 0.1 microgram/ml, 5) DWA showed almost the same ototoxicity as CDDP at the same concentration, 6) Adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium, the ototoxic damage induced by 10 micrograms/ml of KM was not noticed. A protective effect of Ca2+ against KM ototoxicity was observed in vitro. This organ culture and ototoxicity score system can serve as a useful and adequate model system for evaluating the ototoxicity.     

豚鼠壶腹嵴毛细胞内钙离子活动及ATP的影响

目的 探讨离体前庭毛细胞（ＶＨＣ）去极化时细胞内钙离子活动特征及内耳活性物质ＡＴＰ对离体ＶＨＣ内钙离子浓度的影响及其机制。方法 采用酶孵育后机械分离法分离豚鼠壶腹嵴ＶＨＣ,６μｍｏｌ／Ｌ钙荧光探针Ｆｌｕｏ－３染色,利用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下、加入高Ｋ＾＋液及不同浓度ＡＴＰ时代表ＶＨＣ内［Ｃａ＾２＋ｉ的荧光强度相对值的变化。结果 细胞外有Ｃａ＾２＋存在时,７５ｎｍｏｌ／ＬＫ＾     

Variations in receptor cell innervation in the saccule of a teleost fish ear

Using transmission electron microscopy, we quantitatively analyzed the afferent and efferent synapses on 67 sensory hair cells along the saccular epithelium of the oscar (Astronotus ocellatus), a cichild fish with a non-specialized ear. The synaptic profile (number of afferent and efferent synapses per cell) varied considerably among cells. The number of synapses per hair cell ranged from three to 24, and all but six of the 67 hair cells had both afferent and efferent synapses. Statistical analysis showed that the synaptic profiles did not significantly vary anywhere on the saccular epithelium except at the edges. There, hair cells had significantly fewer efferent synapses than hair cells in other epithelial regions. This statistical variation in efferent synapse distribution in different epithelial regions corresponds with the lengths of ciliary bundles in these regions. The synapses on hair cells showed a regional specificity in position. In all cells synapses were never located more apically than the top of the nucleus. On hair cells towards the periphery, the most apical synapse on the hair cells tended to be afferent. On more centrally located cells, the most apical synapse was efferent in 92% of the cells.     

Computer simulation of the mechanical stimulation of the saccular membrane of bullfrog

A three-dimensional computer simulation of the experiment (Benser M. E., Issa N.P., Hudspeth A.J., 1993. Hear. Res. 68, 243-252), devoted to the mechanical stimulation of the surface of saccular membrane of bullfrog with the otoconial mass removed was carried out by finite-element method. Comparison of the calculated distribution of the membrane displacements with the experimental data indicates that the gel layer of the saccular membrane is inhomogeneous. Its lower, thin (6-10 microm) sublayer bordering the macular surface has the smaller Young's modulus which is 20 times less than the modulus of the upper part of the gel membrane (2.5x10(2) N/m(2) and 6.6x10(3) N/m(2), respectively). Possible consequences of this result and modification of the experiment are being discussed. The estimates based on the results of simulation support the conclusion that hair bundle stiffness may dominate the elastic reactance to otolithic membrane shear (Benser et al., 1993).     

Potassium-depolarization induces motility in isolated outer hair cells by an osmotic mechanism

Outer hair cells in vitro contract in response to various stimuli: electrical stimulation, K+-depolarization, elevation of intracellular calcium or osmotic changes of the extracellular medium. The characteristics of motile responses induced by K+-depolarization, osmotic changes, and calcium injection were compared in this study in order to delineate the underlying mechanisms. Slow shape changes in outer hair cells were induced by changes of the osmolality or the K+/Na+-ratio of the bathing medium, or by intracellular injections of calcium. K+- and osmotically induced contractions of isolated outer hair cells had identical morphological features and the same rate (50-200 nm/s) and amplitude (up to greater than 10% of original length) of shortening. The shortening of the cells was linearly related to an increase in volume in both cases. In contrast, the active contraction induced by Ca2+/ATP exhibited a somewhat faster rate and no increase in volume. Furthermore, the K+-induced contractions in outer hair cells, unlike those reported in smooth muscle cells, were unaffected by the removal of external Ca2+ (i.e. medium without Ca2+/Mg2+ and supplemented with 1 mM EGTA) or the presence of D600, an inhibitor of the Ca2+ inward current. The results strongly suggest that K+ induces shape changes of outer hair cells via osmotic forces and that intracellular calcium mediates contractions by a different mechanism.     

Growth of a fish ear: 1. Quantitative analysis of hair cell and ganglion cell proliferation

Proliferation (or addition) of inner ear sensory hair cells continues for a long time postembryonically in cartilaginous and bony fishes, and in amphibians. In contrast, proliferation only occurs during embryonic development in birds and mammals. However, detailed quantitative data on hair cell addition are not available for bony fishes. In order to quantify the extent of proliferation, we determined the number of sensory hair cells on the saccular sensory epithelium in specimens of the cichlid fish Astronotus ocellatus (the oscar) ranging from 2.0 to 19.0 cm in standard length (0.9-343 g). Ganglion cells were counted using serial sections of the saccular branch of the eighth nerve in animals of the same size range. The saccular macula of a 2.0 cm long (0.9 g) Astronotus contains approximately 5500 sensory hair cells; fish from 16 to 19 cm long have over 170 000 hair cells. The increase in number of sensory cells and the increase in both length and weight of the animals studied were statistically correlated (r2 = 0.8). The relative densities of saccular sensory cells in different epithelial regions remained constant in animals from 2.0 to 17 cm; in larger animals the cell density decreased somewhat. Based upon very conservative estimates of the rate of growth of Astronotus, we calculate that an average of 167 hair cells/day are added during the time when the cell population of the saccule increases. Ganglion cell number also increased approximately 4.8 times in the range of fish studied. The smallest animals in our study had about 150 ganglion cells per saccular epithelium, while the largest fish had over 600 ganglion cells. We estimate that the average ratio of hair cells to afferent fibers increases from about 30:1 in the smallest fish to over 300:1 in the largest animals.     

Calcium distribution and mobilization during depolarization in single cochlear hair cells. Imaging microscopy and fura-2

Intracellular distribution of cytoplasm-free Ca2+ concentrations ((Ca2+)i) and dynamic changes during stimulation of viable hair cells were studied using digital imaging microscopy and the Ca2(+)-sensitive dye fura-2. (Ca2+)i was visualized on pseudo-colour images and three-dimensional computer graphics. In the resting state, the intra-cellular distribution of (Ca2+)i in both the outer and inner hair cells was heterogeneous, and the amount of (Ca2+)i in most of the peripheral cytoplasm just beneath the plasma membrane was greater than that throughout the entire cytoplasm. Cell depolarization, induced by elevated K+, led to an increase in (Ca2+)i in the outer hair cells. The increase in (Ca2+)i was not observed under conditions of depolarization in Ca2(+)-free medium. These observations are interpreted to mean that the increase in (Ca2+)i is induced by depolarization with the result that there is an influx of extracellular Ca2+ into the cytoplasm. When Mn2+ was applied during depolarization, a fluorescence quenching occurred. By such means the site of Ca2+ channels was elucidated.     

Slow motility in hair cells of the frog amphibian papilla: Ca2+-dependent shape changes

We investigated the process of slow motility in non-mammalian auditory hair cells by recording the time course of shape change in hair cells of the frog amphibian papilla. The tall hair cells in the rostral segment of this organ, reported to be the sole recipients of efferent innervation, were found to shorten in response to an increase in the concentration of the intracellular free calcium. These shortenings are composed of two partially-overlapping phases: an initial rapid iso-volumetric contraction, followed by a slower length decrease accompanied with swelling. It is possible to unmask the iso-volumetric contraction by delaying the cell swelling with the help of K+ or Cl- channel inhibitors, quinine or furosemide. Furthermore, it appears that the longitudinal contraction in these cells is Ca2+-calmodulin-dependent: in the presence of W-7, a calmodulin inhibitor, only a slow, swelling phase could be observed. These findings suggest that amphibian rostral AP hair cells resemble their mammalian counterparts in expressing both a Ca2+-calmodulin-dependent contractile structure and an "osmotic" mechanism capable of mediating length change in response to extracellular stimuli. Such a mechanism might be utilized by the efferent neurotransmitters for adaptive modulation of mechano-electrical transduction, sensitivity enhancement, frequency selectivity, and protection against over-stimulation.     

A fine method of evaluating the physiological function of vestibular hair cells

We have established a fine method of evaluating the physiological function of vestibular hair cells. We examined the mechano-electrical transduction (MET) to study the physiological function of a hair cell. Hair cells composing the vestibular maculae were loaded with fura-2 AM, and the Ca2+ responses to the mechanical stimulation of hair bundles were monitored. The vestibular maculae were dissected from 1 to 3-day-old rats and were incubated with fura-2 AM for 30 min. Next, the Ca2+ responses generated through the activation of the MET were studied by the water puff stimulation of hair bundles. In addition, the acute effect of gentamicin was confirmed by this method.     

Vestibular evoked myogenic potentials are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs

This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 +/- 1.2/4000 microm(2) in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 +/- 1.8/4000 microm(2)). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.