Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.     

Mechanical properties of sensory and supporting cells in the organ of Corti of the guinea pig cochlea--study by atomic force microscopy

Mammalian hearing is refined by amplification of the motion of the cochlear partition. To understand the cochlear amplification, mechanical models of the cochlea have been used. When the dynamic behavior of the cochlea is analyzed by a model, elastic properties of the cells in the organ of Corti must be determined in advance. Recently, elastic properties of outer hair cells (OHCs) and pillar cells have been elucidated. However, those of other cells have not yet been clarified. Therefore, in this study, using an atomic force microscope (AFM), elastic properties of Hensen's cells, Deiters' cells and inner hair cells (IHCs) in the apical turn and those in the basal and second turns were estimated. As a result, slopes indicative of cell elastic properties were (8.9 +/- 5.8) x 10(3) m(-1) for Hensen's cells (n = 30), (5.5 +/- 5.3) x 10(3) m(-1) for Deiters' cells (n = 20) and (3.8 +/- 2.6) x 10(3) m(-1) for IHCs (n = 20), and Young's modulus were 0.69 +/- 0.45 kPa for Hensen's cells and 0.29 +/- 0.20 kPa for IHCs. There was no significant difference between elastic properties of each type of cell in the apical turn and those in the basal and second turns. However, it was found that there is a significant difference between Young's moduli of cells estimated in this study and those of the OHCs and pillar cells reported previously.     

Structural relationships of the unfixed tectorial membrane

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.     

Characterization of the outer hair cell''s lateral wall membranes

We examined the properties of outer hair cell (OHC) lateral wall membranes by application of 2 fluorescent membrane probes. The markers, C₆-NBD-Ceramide and DiOC₆, have been used in other cell types to label Golgi apparatus and endoplasmic reticulum, respectively. In living isolated OHCs NBD-Ceramide demonstrated uninterrupted fluorescence along the OHC lateral wall, while DiOC₆, labeling proved punctate and notably less uniform in this region. In aldehyde-fixed isolated OHCs both probes exhibited distinct, continuous lateral wall fluorescence. Fixed preparations of the organ of Corti labeled with each probe demonstrated diffuse fluorescence throughout the inner hair cell cytoplasm unlike the uniform, circumferential lateral wall fluorescence seen in OHCs. OHCs exposed to salicylate following NBD-Ceramide labeling displayed patchy, less distinct labeling along the OHC lateral wall. The thickness of lateral wall fluorescence in salicylate exposed cells was 49% greater than control OHCs. We interpreted the salicylate induced change in lateral wall labeling as a fluorescent representation of previously described ultrastructural dilatation and vesiculation of the subsurface cisternae. The distribution of these 2 fluorescent probes along OHC lateral wall membranes suggests that the OHC's subsurface cisternae are neither Golgi nor ER, but share characteristics of both.     

TRPA1-Mediated Accumulation of Aminoglycosides in Mouse Cochlear Outer Hair Cells

Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin–Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.     

体外培养小鼠耳蜗Corti器对庆大霉素的吸收

目的 观察庆大霉素-德州红耦联物在体外培养小鼠耳蜗Corti器的分布情况,比较不同时间庆大霉素吸收的差异.方法 分离小鼠耳蜗Corti器,体外培养,培养液中加有庆大霉素-德州红耦联物,分别于培养后3 h、6 h、12 h、24 h、48 h、3 d.4 d和7 d收集标本,荧光染色后行激光扫描共聚焦显微镜观察基底膜庆大霉素的分布情况.结果 小鼠耳蜗Corti器在培养3 h后即可见外毛细胞的纤毛和胞体两侧的胞膜有庆大霉素分布;随着培养时间的延长,庆大霉素在Corti器中的所有细胞均见分布,尤以外毛细胞明显,主要聚集在毛细胞顶端纤毛的下方;培养24 h后庆大霉素在外毛细胞的聚集达到最高峰,而在支持细胞未见明显的聚集;体外培养7 d后在毛细胞胞质中仍可见庆大霉素分布.结论 小鼠Corti器体外培养可用来动态检测庆大霉素在耳蜗细胞的吸收和聚集情况.     

庆大霉素鼓室内注射后在内耳细胞中的分布

目的 观察鼓室内注射庆大霉素后，不同时间庆大霉素在前庭和耳蜗中的分布。方法 将庆大霉素同德州红连接形成庆大霉素-德州红耦联物后，行豚鼠鼓室内注射，注射后12h，1、2、3、4、7、14、28d处死动物，Phalloidin染色后运用激光共聚焦扫描显微镜观察基底膜、椭圆囊、球囊、外半规管壶腹嵴庆大霉素分布情况，并进行荧光分布半定量分析。结果 庆大霉素自注射后12h起在内耳所有细胞均见分布，在基底膜的外毛细胞、椭圆囊、球囊、外半规管壶腹嵴的感觉细胞聚集明显，主要聚集在毛细胞顶端纤毛下方的细胞质中，支持细胞分布较少。注射后第3天庆大霉素在内耳聚集达到最高峰，并在毛细胞内聚集较长时间。结论 庆大霉素-德州红耦联物是一个研究庆大霉素在内耳分布的良好的荧光探针，可用来检测庆大霉素的药代动力学和聚集机制．     

Differences in the distribution of F-actin in outer hair cells along the organ of Corti

There is evidence of differences in the structure, innervation and physiological responses between outer hair cells (OHCs) of the basal and apical turns of the mammalian cochlea. In this study we have used rhodamine-labelled phalloidin to investigate the differential distribution of F-actin in OHCs along the organ of Corti of the guinea pig. Isolated OHCs and surface preparations and cryosections of the organ of Corti were studied. F-actin was observed in stereocilia and the cuticular plate of all OHCs. In addition, some OHCs had a network of F-actin extending from the cuticular plate towards the nucleus. This infracuticular network was observed in most OHCs of the apical cochlear turns but was not seen in any OHCs of the basal turn. These microstructural differences between OHCs of the base and apex could be related to differences in OHC function between the apical and basal portions of the cochlea.     

Noise exposure induced cochlear hair cell death pathways in guinea pig

Objective To understand the mechanism of noise exposure induced outer hair cells(OHCs) death pathways. Methods Thirty two guinea pigs were used in this study. The animals were either exposed for 4 h/day to broadband noise at 122 dB SPL (A-weighted) for 2 consecutive days or perfused with MNNG. After auditory test,the cochleae of animals were dissected. Propidium iodide (PI), a DNA intercalating fluorescent probe, was used to trace morphological changes in OHC nuclei. F-actin staining was used to determine missing OHCs. Caspase-3 was detected in living organ of Corti whole mounts using the fluorescent probe. The single strand DNA (ssDNA) in apoptotie OHCs in guinea pigs and apoptosis inducing factor (AIF) in hair cells in guinea pigs were examined by immunohistology method. Whole mounts of organ of Corti were prepared. Morphological and fluorescent changes were examined under a confocal microscope. Results (1) Both apoptotic and necrotic hair cells appeared following noise exposure. (2) Noise exposure induced single strand DNA in apoptotic OHCs but not in the normal OHCs. (3)Either after noise exposure or after MNNG perfusion, apoptotic OHCs were featured by nuclear condensation or fragmentation with caspase-3 activation, whereas necrotic OHCs were characterized by nuclear swelling without caspase-3 activation. (4) In normal organ of Corti, AIF was located in the mitochondria areas. After noise exposure,AIF was translocated from mitochondria in apoptotic and necrotic OHCs. Conclusion These findings indicate that noise exposure damages DNA in the OHC, which triggers action of Caspase-3. Subsequently, AIF is translocated to the nucleus, leading to DNA damage and OHCs death.     

豚鼠耳蜗毛细胞丝状肌动蛋白表达

目的研究正常生理状态下丝状肌动蛋白(F-actin)的结构特征和分布规律。方法应用免疫细胞化学方法,采用单克隆抗体及免疫荧光标记技术,借助荧光显微镜和激光扫描共聚焦显微镜,逐层观察耳蜗毛细胞骨架蛋白-丝状肌动蛋白的分布特点。结果在连续的光学切片上,可以看到静纤毛、表皮板和外毛细胞(outerhaircells,OHCs)的周围环都有明亮的鬼笔环肽染色。除基底转外,其余各转皮板下网络(infracuticularnetwork,ICN)均有鬼笔环肽显色,外毛细胞侧壁着色亮度自基底转到顶转逐渐减弱。内、外柱细胞和Deiters细胞染色明亮,其中耳蜗各转外柱细胞头板的显色亮度都强于OHCs。结论豚鼠耳蜗OHCs中F-actin的结构和分布存在差异,内毛细胞(innerhaircells,IHCs)的结构和分布无差异,表明局部结构的梯度决定局部功能的差异。     

Effects of intense noise exposure on the outer hair cell plasma membrane fluidity

Outer hair cells (OHCs) play an important role in cochlear amplification via their length changes (electromotility). A noise-induced cochlear amplification loss leading to a permanent threshold shift (PTS) was observed without a significant hair cell loss in rats [Chen, G.D., Liu, Y., 2005. Mechanisms of noise-induced hearing loss potentiation by hypoxia. Hear. Res. 200, 1-9.]. Since motor proteins are inserted in the OHC lateral membrane, any change in the OHC plasma membrane may result in a loss of OHC electromotility, leading to a loss of cochlear amplification. In this study, the lateral diffusion in the OHC plasma membrane was determined in vitro in guinea pigs by fluorescent recovery after photobleaching (FRAP) after an in vivo noise exposure. The lateral diffusion in the OHC plasma membrane demonstrated a length-dependence, which increased as OHC length increased. A reduction in the lateral diffusion was observed in those OHCs with lengths of 50-70 microm after exposure to an 8-kHz octave band noise at 110 dB SPL for 3h. This membrane fluidity change was associated with the selective PTS at frequencies around 8 kHz. The reduction of the lateral diffusion in the OHC lateral wall indicated that noise could impair the micromechanics of the OHC lateral wall and might consequently impair OHC electromotility to induce threshold shift.     

Functional Prestin Transduction of Immature Outer Hair Cells from Normal and Prestin-Null Mice

Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.     

THE COCHLEAR AMPLIFIER： IS IT HAIR BUNDLE MOTION OF OUTER HAIR CELLS？

Cochlear outer hair cells （OHCs） are involved in a mechanical feedback loop in which the fast somatic motility of OHCs is required for cochlear amplification. Alternatively, amplification is thought to arise from active hair bundle movements ob- served in non-mammalian hair cells. We measured the voltage-evoked hair bundle motions in the gerbil cochlea to determine if such movements are also present in mammalian OHCs. The OHCs displayed a large hair bundle movement that was not based on mechanotransducer channels but based on somatic motility. Significantly, bundle movements were able to generate radial motion of the rectorial membrane in situ. This result implies that the motility-associated hair bundle motion may be part of the cochlear amplifier.     

豚鼠耳蜗各回外毛细胞L型钙通道的分布

目的 探讨耳蜗2－4回外毛细胞（outer hair cell,OHC)L型钙通道分布密度。方法 豚鼠耳蜗外毛细胞L型钙通道经DM－BODIPY DHP和RH414标记后用激光共聚焦显微镜观察，以DM－BODIPY DHP／RH414荧光比值大小指示L型钙通道密度的高低。结果 （1）应用硝苯地平阻断后，质膜的DM－BODIPY DHP荧光染色明显减弱，表明其钙通道为特异性L型钙通道。（2）L型钙通道密度密度从低频区（第4回）向高频区（第2回）有逐渐增加的趋势，方差分析示第4回和第2回OHC之间L型钙通道的分布差异有显著性意义；（3）OHC质膜不同区域L型钙通道分布差异无显著性意义。结论 耳蜗2－4回OHC之间L型钙通道分布的差异可能是耳蜗音位分布图的分子机制之一。     

豚鼠耳蜗各回外毛细胞L型钙通道的分布

目的探讨耳蜗2～4回外毛细胞(outer hair cell,OHC)L型钙通道分布密度.方法豚鼠耳蜗外毛细胞L型钙通道经DM-BODIPY DHP和RH414标记后用激光扫描共聚焦显微镜观察,以DM-BODIPY DHP / RH414荧光比值大小指示L型钙通道密度的高低.结果①应用硝苯地平阻断后,质膜的DM-BODIPY DHP荧光染色明显减弱,表明其钙通道为特异性L型钙通道;②L型钙通道密度从低频区(第4回)向高频区(第2回)有逐渐增加的趋势,方差分析示第4回和第2回OHC之间L型钙通道的分布差异有显著性意义;③OHC质膜不同区域L型钙通道分布差异无显著性意义.结论耳蜗2～4回OHC之间L型钙通道分布的差异可能是耳蜗音位分布图的分子机制之一.     

Succinic dehydrogenase histochemistry as an early marker for hair cell pathology

Density measurements of succinic dehydrogenase (SDH) activity were obtained from the inner and outer hair cells on surface preparations obtained from the guinea pig cochlea. Guinea pigs were exposed to noise (3.85 kHz, 120 dB SPL, 22.5 min) and sacrificed 0, 4 or 24 h after the exposure. By 4 h after exposure, the first- and second-row outer hair cells already demonstrated an altered SDH activity. By 24 h after exposure, a significant decrease in SDH staining in both the inner and outer hair cells at a distance of 10-12 mm from the cochlear apex was demonstrated. After a 1-month recovery period, scanning electron microscopy confirmed the main lesion site to be at a distance of 10-12 mm. In addition, Hensen's cells (supporting cells) at a distance of 10-12 mm from the apex were intensely stained by SDH after noise exposure, indicating an increase in oxidative metabolism. SDH staining in the Hensen's cells from the unexposed cochleae was not found. In conclusion, our findings suggest that the early use of SDH histochemistry can predict later permanent damage to the organ of Corti.     

硫酸链霉素引起的大鼠体外培养耳蜗毛细胞凋亡

目的 探讨在离体培养条件下硫酸链霉素是否通过半胱氨酸天冬氨酸蛋白酶(caspase)的激活而导致大鼠耳蜗毛细胞凋亡.方法 将出生后3～4 d的大鼠耳蜗基底膜取出进行离体培养,培养过夜后用0.1 mmol/L硫酸链霉素培养液继续培养24 h.终止培养前1 h应用碳氧荧光素标记的肽荧光甲基酮(carboxyfluorescein labeled peptide fluoromethyl ketone,FAM-peptide-FMK)标记的caspase-6,caspase-8,或caspase-9指示剂作用1 h,然后应用异硫氰酸四甲基罗丹明(tetramethyl rhodamine iso-thiocyanate,TRITC)标记的鬼笔环肽(phalloidin)标记毛细胞的纤毛和表皮板,再应用Topro-3 DNA探针对细胞核进行染色,最后在共聚焦显微镜下观察.结果 1 mmol/L硫酸链霉素引起的耳蜗毛细胞缺失在底回约为80%,在顶回约为20%.经链霉素作用后,耳蜗毛细胞的细胞核发生浓缩和碎裂,同时可见cmpase-6,caspase-8和caspase-9阳性表达.结论 硫酸链霉素可以引起离体培养的大鼠耳蜗毛细胞凋亡,上游和下游的caspase激活可能是其主要途径.     

Nitric oxide distribution and production in the guinea pig cochlea

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.     

Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility in zebrafish (Danio rerio)

Developmental differences in hair cell susceptibility to aminoglycoside-induced cell death has been observed in multiple species. Increased sensitivity to aminoglycosides has been temporally correlated with the onset of mechanotransduction-dependent activity. We have used in vivo fluorescent vital dye markers to further investigate the determinants of aminoglycoside induced hair cell death in the lateral line of zebrafish (Danio rerio). Labeling hair cells of the lateral line in vivo with the dyes FM 1-43, To-Pro-3, and Yo-Pro-1 served as reliable indicators of hair cell viability. Results indicate that hair cell maturation is a determinant of developmental differences in susceptibility. The age dependent differences in susceptibility to aminoglycosides are independent of the onset of mechanotransduction-dependent activity as measured by FM 1-43 uptake and independent of hair cell ability to take up fluorescently conjugated aminoglycosides.