Membrane specializations in the human organ of Corti

The human organ of Corti was investigated with the freeze fracturing technique with the purpose of analysing membrane specializations. Tight junctions were found on hair cells as well as on supporting cells. Inner and outer hair cells were coupled to the supporting cells by rather extensive tight junctions. The tight junctions between the Deiter's cells were comparable to those of the hair cells, while the tight junctions between the Hensen's cells were considerably less extensive. Gap junctions were present coupling all supporting elements in the organ of Corti, small ones preferably in the apical regions of the cells and large ones in the basal region.     

Intercellular communication in the supporting cells of the organ of Corti

We have directly tested the concept that the supporting cells of the organ of Corti are functionally coupled through gap junctions. In vitro and in vivo preparations were evaluated. Electrical measurements clearly show that the cells are coupled ionically. Voltage drops measured in neighboring cells in response to intracellular current injections indicate that current spread decays rapidly. Despite the existence of electrical coupling, fluorescent dye injection studies revealed no dye spread into adjacent cells, other than a few instances which were clearly artifactual. However, it is possible that dye spread is very slow and that dye in adjacent cells is diluted below visual detectability. In any case, dye coupling is remarkably poor compared to other electrically coupled tissues. The role of coupling in the supporting cells may be nutritive, considering the avascular nature of Corti's organ.     

Hensen's cell cyst of the organ of Corti

The temporal bone collection at the Massachusetts Eye and Ear Infirmary contains the bones of two subjects with bilaterally symmetrical cyst formations in the Hensen's cell areas of the organs of Corti. In both subjects the cysts are located in the 8-14-mm regions of the cochlear ducts. Both cochleas of one subject show pressure atrophy of the outer hair cells, demonstrating that the cysts may have the potential for producing high-tone hearing loss. While the genesis and contents of the cysts are unknown, it is postulated that they may represent the coalescence of lipid droplets extruded from Hensen's cells.     

Glycocalyx heterogeneity in normal and hydropic cochleas of the guinea pig.

The endo- and perilymphatic glycocalyx of the cochlear epithelia were investigated ultrastructurally in normal and hydropic cochleas using the electron-dense markers cationized ferritin and colloidal thorium. In the normal cochleas all epithelial surfaces showed reactivity with both markers. Using cationized ferritin, no regional differences in reactivity could be demonstrated. With colloidal thorium, however, the apical membranes of the outer hair cells reacted more intensely than either the basolateral membranes or the membranes of the supporting cells of the organ of Corti. Cationized ferritin reactivity was not affected by digestion with either neuraminidase or hyaluronidase. In contrast, colloidal thorium reactivity of the endolymphatic surfaces was greatly reduced after neuraminidase digestion. Reactivity of the cell membranes lining the perilymphatic compartments of the cochlea was less affected by neuraminidase digestion, except for the basolateral membranes of the sensory and supporting cells of the organ of Corti, which demonstrated a greatly reduced reactivity. These findings indicate that the glycocalyx of the epithelial surfaces lining the endolymphatic compartment has a high content of sialic acid. The differences that are observed in normal cochleas in regard to colloidal thorium reactivity between the apical membranes of the outer hair cells and the membranes of the Deiters' cells, could not be demonstrated in hydropic cochleas. This probably contributes to the early functional changes in outer hair cells observed in experimental hydrops.     

Ultrahistochemical demonstration of glucose-6-phosphatase in hair cells of the guinea pig organ of corti (author's transl)]

This study is concerned with the ultrahistochemical demonstration of glucose-6-phosphatase activity and its localization in inner and outer hair cells of the guinea pig organ of Corti. The enzyme activity has been demonstrated by a method according to Hugon et al. (1970). In hair cells of the organ of Corti a characteristic distribution pattern of reaction products has been registered. Subsurface cisterns and the Hensen's bodies of outer hair cells were heavily loaded with reaction products. In addition, the endoplasmic reticulum and the nuclear membrane as well as postsynaptic cisterns were rich in precipitates. With regard to their morphologic pecularities the inner hair cells show a more homogeneous distribution of enzyme activity. The findings corroborate the former assumption of a genetic relationship of either subsurface cisterns and Hensen's body to the endoplasmic reticulum of outer hair cells. Furthermore, the high glucose-6-phosphatase activity of both subsurface cisterns and Hensen's bodies are considered indicative of their participitation in the energy metabolism of outer hair cells. Referring to biochemical studies of Thalmann and associates (1973), the narrow spatial relationship of glucose-6-phosphatase positive ER membranes to mitochondria presumably represents a morphologic correlation of aerobic and anaerobic metabolic pathways in the guinea pig organ of Corti.     

Gentamicin-induced alterations of succinic dehydrogenase activity in the organ of Corti as revealed by non-decalcified frozen sections of the guinea pig's cochlea

Summary The distribution of succinic dehydrogenase (SDH) activity and the changes of SDH activity after injection of gentamicin (GM) were observed in the organ of Corti using non-decalcified frozen sections of the guinea pig's cochlea. The distribution of SDH activity was found to increase from the apex to the basal turn. At each turn, SDH activity of the inner hair cells, the inner supporting cells and the nerve endings surrounding the supporting cell and on the hair cells presented a greater activity than that found in the outer hair cells, adjacent Deiter's cells and associated nerve endings. It was further observed that GM had a greater effect on SDH activity in the basal turn than the other turns. At each turn, a more sensitive area of response to GM was found on the nerve endings one each hair cell, especially on the outer hair cells of the basal turn.The experimental part of this study was completed at the Laboratory of Autoradiography, Beijing Medical University, Beijing, People's Republic of China     

豚鼠耳蜗部分外支持细胞的分离法

目的 :探讨豚鼠耳蜗部分外支持细胞 (Deiter细胞、Hensen细胞和外柱细胞 )的分离方法 ,并建立细胞活性的鉴别标准。方法 :选取健康杂色豚鼠 5只 ,解剖出耳蜗基底膜 ,采用酶解加机械吹打法分离Deiter细胞、Hensen细胞和外柱细胞。结果 :可以获得较多数量、活性良好、长短不一的Deiter细胞和Hensen细胞 ,8h内细胞活性较好 ;但外柱细胞数量较少 ,存活时间较短。评价Deiter细胞、Hensen细胞和外柱细胞的活性标准 :①细胞膜无膨胀或扭曲 ;②细胞核无肿胀、移位 ;③在相差显微镜下细胞呈半透明状态 ,存在双折射现象 ;④细胞内无呈布朗运动的颗粒。结论 :熟悉耳蜗的解剖特性、分离出完整的基底膜 ,增加酶的浓度和加大机械吹打力度是获取活性良好的Deiter细胞、Hensen细胞和外柱细胞的关键 ;评价外毛细胞活性的 4项标准同样可用于判断Deiter细胞、Hensen细胞和外柱细胞的活性     

Electrical coupling differs in the in vitro and in vivo organ of Corti

Electrical communication between the supporting cells of the guinea pig organ of Corti was studied. For in vitro experiments, the inner ear was rapidly removed and placed in a heated perfusion chamber. Medium 199 was used. The bony cochlea and the lateral wall (spiral ligament and stria vascularis) were removed to expose the top two coils of the organ of Corti. In vivo experiments were performed upon anesthetized animals whose cochleas were exposed surgically. A tiny fenestra was made in the bony cochlea which permitted the passage of electrodes through the lateral wall and into the organ of Corti of the third turn. Coupling was assessed by impaling neighboring cells with 3 M KCl electrodes, and noting the spread of intracellularly injected current. Coupling ratios in the in vitro preparation were consistently greater than those obtained in vivo (0.58 +/- 0.17 vs. 0.104 +/- 0.064). Differences exist between the in vitro and in vivo preparations which might account for these results. In vivo the supporting cells are bathed in two different media, endolymph apically, and perilymph basally. Consequently, on their apical side the supporting cells are exposed to fluid high in K+, low in Ca2+ and at a potential of 80 mV, the endolymphatic potential. In vitro the cells are bathed on all sides in fluid similar to perilymph. Intermixing the fluids in an in vivo preparation, by tearing away the stria vascularis and Reissner's membrane, increases the magnitude of the coupling ratio (0.455 +/- 0.209). Thus the unique microenvironment of the inner ear maintains lower coupling ratios, and smaller space constants for the supporting cells.     

Expression of intermediate filament proteins in the adult human cochlea.

The immunohistochemical localization of intermediate filament proteins was studied in frozen sections of chemically fixed, nondecalcified adult human cochleas. Cytokeratins were found in all epithelial cells lining the cochlear duct (including most supporting cells of the organ of Corti) but were absent in the hair cells. Neurofilament proteins were present in the nerve endings at the hair cells, in the neural bundles, and in the ganglion cells. Vimentin staining occurred in most of the supporting structures and was roughly complementary to the regions showing cytokeratin staining and neurofilament staining. However, the region of the spiral prominence and outer sulcus, as well as the pillar cells and Deiters' cells in the organ of Corti, showed coexpression of vimentin and cytokeratins. No definite immunostaining was observed with antibodies to desmin and glial fibrillary acidic protein.     

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.     

Degeneration of the organ of Corti following intravenous administration of cisplatin.

The development of acute morphological changes in the cochlea was studied in guinea pigs given one intravenous high-dose injection of cisplatin. In the light microscope three major stages of degeneration in the organ of Corti could be recognized: 1) an initial swelling of the Hensen's cells and protrusion of the Deiters' cells into the space of Nuel enclosing the outer hair cells, 2) a gradual degeneration of the outer hair cells together with a vacuolization in the region of the base of the inner hair cells, 3) a collapse of the Reissner's membrane and the entire organ of Corti with different degrees of damage to the inner hair cells. Sporadic bulging of the marginal cells of the stria vascularis into the endolymphatic space could be observed 4 days after injection.     

Cryoprobe-induced apical lesions in the chinchilla. I. Morphological effects of lesioning parameters

To create experimental lesions localized to the low frequency region of the organ of Corti, a cryoprobe was applied to the apical area of 37 cochleas from 26 adult chinchillas. Twenty cochleas were exposed to single applications of a cryoprobe for 2.5, 3.0 and 3.5 min; 17 cochleas were exposed to two applications of 1.5, 2.0 and 3.0 min each with about a 3 min interval between applications. Cryoprobe tip temperature rose from about -140 degrees C when placed on the apex to about -80 degrees C after a continuous 3.5 min application. Survival time after lesioning was from 2 to 75 days, with most being 12 days or less. All cochleas except one sustained regions of damage characterized by complete absence of the organ of Corti and by missing hair cells indicated by extensive scarring. Inner hair cells were less susceptible to damage than were outer hair cells. Well-defined lesions which were continuous over the apical organ of Corti were found in some cochleas exposed to single probe applications, but such applications more often resulted in lesions which had areas of less damage. Of the various application protocols used, two applications of 1.5 min each, with an interval to allow the tissue to warm, most consistently produced severe and discrete apical lesions. In 9 of 13 cochleas exposed to two 1.5 min probe applications, such lesions extended about 35% or less of the distance from the apex. In most cochleas, regardless of the severity of the apical lesion, the pattern of hair cell rows and stereocilia configuration appeared normal in the basal 40-50% of the organ of Corti.     

The fate of outer hair cells after acoustic or ototoxic insults

In epithelial sheets, clearance of dead cells may occur by one of several routes, including extrusion into the lumen, phagocytic clearance by invading lymphocytes, or phagocytosis by neighboring cells. The fate of dead cochlear outer hair cells is unclear. We investigated the fate of the "corpses" of dead outer hair cells in guinea pigs and mice following drug or noise exposure. We examined whole mounts and plastic sections of normal and lesioned organ of Corti for the presence of prestin, a protein unique to outer hair cells. Supporting cells, which are devoid of prestin in the normal ear, contained clumps of prestin in areas of hair cell loss. The data show that cochlear supporting cells surround the corpses and/or debris of degenerated outer hair cells, and suggest that outer hair cell remains are phagocytosed by supporting cells within the epithelium.     

The effects of cytoplasmic acidification upon electrical coupling in the organ of Corti

The supporting cells of the organ of Corti are joined to one another by gap junctions, and electrical coupling among them is known to be good. It is demonstrated here, using an in vitro preparation, that electrical communication between Hensen's cells can be modified by treatments which are known to cause cytoplasmic acidification. Treatment of the preparation with 100% CO2-saturated medium causes a drop in membrane potential, increase in input resistance, and decrease in steady-state coupling ratio. These measures return to pretreatment levels upon washout of the CO2 medium. Also, direct injection of H+ into a Hensen's cell uncouples that cell from the supporting cell network. An increase in coupling ratio is sometimes observed immediately before and after uncoupling due to CO2 treatment. In fact, in some cases it is possible to solely increase coupling ratios with limited CO2 treatments, although prolonged treatment with CO2 invariably produces uncoupling. This phenomenon may be due to an increase in cell resistance without a change in junctional conductance. A few possible roles for gap junctions in the inner ear are suggested, and the significance of the present results discussed.     

出生后大鼠听毛细胞纤毛发育过程

目的观察新生大鼠（P1、P7、P14、P21）耳蜗Corti器的形态结构及排列方式,以便了解听毛细胞纤毛发育的过程。方法取新乍SD大鼠四个阶段的耳蜗,采刖扫描电镜对出生后大鼠耳蜗Corti器形态结构进行系统观察。结果发现这四个阶段在体犬鼠耳蜗毛细胞从出生至毛细胞完全发育成熟,绝大部分都是以一排内毛细胞和三排外毛细胞的方式排列的,只有少部分在局部发现多出几个外毛细胞形成第四排。毛细胞的形态结构及排列方式随着时间的改变逐渐发育成熟。P14这个阶段是个重要的时期,Corti器表面柱细胞头板增宽,表面的微绒毛减少,Deiter细胞表面微绒毛也减少,动纤毛从底圄至顶圈逐渐退化已经基本结束,P14这个阶段Corti器各个部分发育已经完全成熟：结论出生后14天之内。大鼠听毛细胞静纤毛和动纤毛与听器其他细胞形态结构仍不同程度地处于由不成熟到成熟的逐渐发育过程,为研究大鼠出生后Corti器的发育、听觉功能的建立和完善提供了比较可靠的形态学证据。     

Localization of the P2Y4 receptor in the guinea pig organ of Corti

Cochleae of guinea pigs were evaluated for the presence of the metabotropic receptor, P2Y4. Evidence is presented that P2Y4 protein is expressed in the guinea pig cochleae using Western blot analysis. A single protein band of 35 kDa was detected with P2Y4 receptor-specific antibody. The cellular distribution of P2Y4 purinoceptor protein was determined by immunohistochemistry of the whole organ of Corti. Immunoreactive staining for P2Y4 was seen in most cells of the organ of Corti. Staining of Hensen's cells and Deiters' cells, especially the outer Deiters' cells, was more intense than staining of the outer hair cells, inner hair cells, and pillar cells. Staining intensity was greatest at the basal turn and progressively decreased in the upper turns with the apex showing the weakest staining pattern. This is the first demonstration of a metabotropic P2Y receptor in the guinea pig organ of Corti.     

Experimental mumps labyrinthitis in monkeys (Macaca irus)--immunohistochemical and ultrastructural studies

Three monkeys (Macaca irus) were inoculated with mumps virus into unilateral cochleas and their inner ear were examined by immunofluorescent microscopy and transmission electronmicroscopy. The temporal bones were removed after survival period of 14 days when serological tests disclosed elevation of anti-mumps antibody titers. Immunofluorescent microscopy revealed that the viral antigen was positive in the stria vascularis. The ultrastructural study revealed that the pathologic changes in the cochleas were marked in the organ of Corti and stria vascularis. The outer hair cells were more susceptible to the infection than the inner hair cells. In the stria vascularis, both marginal and intermediate cells were affected. It was possible to find some of marginal cells in the basal turn shedding a large number of mature virions into the endolymph. These pathologic changes observed in the cochleas of the monkeys were similar to those previously revealed in the guinea pig cochleas and thus were considered as the specific features of acute mumps labyrinthitis.     

Ultrastructural damage in cochleas used for studies of basilar membrane mechanics

Cat cochleas used for interferometric studies of basilar membrane mechanics were examined with the electron microscope. The structures most severely damaged in the experimental cochleas are the outer hair cells and the radial afferent fibers to the inner hair cells. Since the basilar membrane and other supporting structures appear to be normal, mechanical changes observed in the experimental cochleas are most probably due to outer hair cell damage. Individual animals with varying degrees of damage showed large differences in the frequency of basilar membrane resonance at the same place in the cochlea. Shifts in tuning of this magnitude could occur as a consequence of hair cell damage only if the stiffness of the stereocilia and associated structures was greater initially than the stiffness of the basilar membrane and gradually decreased with damage. The present series of observations, therefore, suggest that the stiffness of the outer hair cell stereocilia determines basilar membrane tuning.     

Actin-binding and microtubule-associated proteins in the organ of Corti.

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.     

A newly identified surface coat on cochlear hair cells

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.