Immunohistochemical identification and localization of actin and fimbrin in vestibular hair cells in the normal guinea pig and in a strain of the waltzing guinea pig

Using immunohistochemical methods, actin and fimbrin were identified and localized in vestibular epithelia iin the normal guinea pig and in the waltzing guinea pig. In the normal guinea pig, actin was found in the stereocilia and in the cuticular plate. Fimbrin was detected in the stereocilia but surprisingly not in the cuticular plate. As fimbrin was found in the cuticular plates in cochlear hair cells it is suggested that the hair cells in the two organs have different mechanoreceptor properties which can demand different cuticular plate stability. In the waltzing guinea pig, actin was found in the stereocilia, in the rod and n the cuticular plate. Fimbrin was seen in the stereocilia and in the rod, but could not be detected in the cuticular plate. These results emphasize the resemblance between the rod and the stereocilia. It is suggested that the rod is a genetically induced pathological intracellular type of stereocilium which grows in an uncontrolled manner.     

Three-dimensional visualizations of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membranous organelles

The fine structure of the filamentous and membranous organelles in the stereocilia and in the cuticular plate of sensory hair cells from the guinea pig was examined using a rapid-freeze, deep-etch method. In fixed and unfixed tissue the outer surface of the plasma membrane of the stereocilia had numerous surface protrusions of various sizes and shapes, while the protoplasmic fractured face of the membrane had rather sparse intramembrane particles. Many tiny cross links were present between the adjacent actin filaments and between actin filaments and the plasma membrane of the stereocilia. Numerous fibrils radiating from the hair rootlet were attached to the peripheral actin filaments in the cuticular plate. The radiating fibrils differed from the tiny cross links which interconnected the adjacent, randomly-oriented actin filaments in the cuticular plate. These complex structures consisting of actin filaments in the hair rootlets, radiating fibrils, and peripheral actin filaments may play an important role in regulating stereociliary bending.     

Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats

Semithin sections (1 micron) of human fetuses and young cat vestibular epithelium embedded in Epon were reacted with primary antibodies raised against myosin, tropomyosin, and actin. The results were very similar in the two species. Myosin and tropomyosin were colocalized and strictly limited to an apical superficial corona arranged in a punctiform pattern. The stereocilia and cuticular plate were unreactive to myosin and tropomyosin antibodies. Actin antibodies stained the stereocilia, cuticular plate, and a marginal ring surrounding and underlying the cuticular plate. At this level, myosin and tropomyosin were not detected. This result suggests that the circumferential actin ring has a structural role. Colocalization of myosin, tropomyosin, and actin in a superficial area around the stereocilia bundle and along the apical surface of the hair cell imply that active processes could exist at the apex of certain sensory vestibular hair cells. The specificity of myosin antibodies and their cross-reactivity with different types of myosin are discussed.     

Chronic ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates

Two cochleas with permanent, noise-induced threshold shifts of 40 to 60 dB (as measured by responses of single, auditory-nerve fibers) were analyzed in detail, first at the light-microscopic level, and subsequently with transmission electron microscopy of serial sections. The light-microscopic analysis showed that there was little hair cell loss, but widespread damage to the stereocilia, especially those on the inner hair cells and first-row outer hair cells. Transmission electron microscopy revealed no pathology in any cells or organelles in the organ of Corti except for the stereocilia and their rootlets. Stereocilia tufts on first-row OHCs and IHCs were badly damaged; those on second- and third-row OHCs appeared ultrastructurally normal. Within the IHC tuft, the damage to the tall, outer row of stereocilia was often selective: the shorter rows could remain ultrastructurally normal even when the tall row was completely missing. The data suggested that most of the structures which appear normal in a careful light-microscopic analysis are also normal at the ultrastructural level. This strengthens earlier suggestions that the correlations between light-microscopic stereocilia changes and alterations in single-unit physiology are causal in nature. The most common stereocilia pathologies were fracture, attenuation or complete loss of the stereociliary rootlets, especially in the region of the cuticular plate near the endolymphatic surface of the cell. The degree and extent of these changes were well correlated with the degree and extent of stereocilia disarray. Abnormalities of the actin-filament matrix within the stereocilia were extremely rare in unfused stereocilia, however, they were common when the stereocilia were part of a fusion bundle. Fusion of stereocilia was always associated with ectopic supracuticular cytoplasm. Based on the ultrastructural observations, different sequences of structural changes preceding the generation of disarray, loss or fusion of stereocilia are suggested.     

Preservation and visualization of actin-containing filaments in the apical zone of cochlear sensory cells

The fine filamentous structure in the apical zone of cochlear sensory cells of the guinea pig was investigated under transmission electron microscopy (TEM) using various fixation methods. The true form of this structure, which is that of a dense core of sensory hairs and cuticular plates containing hair rootlets, has been hitherto unknown because of the selectively destructive effect of ordinary fixatives. We revealed the fine filamentous structure in great detail by fixing the specimens in tannic acid or by the modified glutaraldehyde-osmium fixation method, which can preserve actin filaments during the procedures required to prepare the specimen for TEM. The filamentous structure gives the impression of a negatively stained image when prepared in this way. Filaments were packed regularly and tightly into dense cores which projected down deep into the cuticular plate as hair rootlets. Cross-striations were seen at intervals of 360 ± 28 Å along the packed filaments, a distance which is comparable to the periodicity of an actin paracrystal. The overall diameter of each filament was 83 Å. In fact, the structure of dense cores and hair rootlets proved to be composed of actin paracrystals, probably containing some regulatory proteins. Cross-sectioned actin filaments in the paracrystal were arranged in an extremely regular hexagonal pattern. The characteristic filamentous texture in the cuticular plate was best seen in tissues that were pretreated with EDTA, and then fixed by tannic acid. It is probable that the greater part of the cuticular plate is composed of actin filaments and actin monomers, both containing Ca²⁺-dependent regulatory proteins. Utilizing the above ultrastructural findings, some functional models of this zone are proposed.     

Mechanisms of movement in outer hair cells and a possible structural basis

Summary Isolated outer hair cells were found to slowly shorten when subjected to a solution that would induce contraction in a muscle fibre. Two possible mechanisms underlying this behaviour emerge from ultrastructural and immunocytochemical investigations. Antibody labelling at the electron microscopic level demonstrates that actin is present not only in the stereocilia and in the cuticular plate but also along the wall of outer hair cells, between the plasma membrane and the subsurface fenestrated cisternae. The latter are interconnected by regularly spaced pillars, resembling those seen between the T-tubules and sarcoplasmic reticulum in muscle fibres. Contraction also results from the application of positively charged macromolecules to the bathing solution. This implies sensitivity of the membrane-associated complex (the cortex system) to an electrical current. A second contractile system may reside in the cytoplasm, where calmodulin is present in contracted hair cells. This protein is a calcium-binding control protein for contraction-like events in smooth muscle and non-muscle cells. The unique presence of the cortex system in outer hair cells, and its absence in inner hair cells, indicates a functional significance that relates to a motor function of outer hair cells in hearing.     

Mechanisms of movement in outer hair cells and a possible structural basis

Isolated outer hair cells were found to slowly shorten when subjected to a solution that would induce contraction in a muscle fibre. Two possible mechanisms underlying this behaviour emerge from ultrastructural and immunocytochemical investigations. Antibody labelling at the electron microscopic level demonstrates that actin is present not only in the stereocilia and in the cuticular plate but also along the wall of outer hair cells, between the plasma membrane and the subsurface fenestrated cisternae. The latter are interconnected by regularly spaced pillars, resembling those seen between the T-tubules and sarcoplasmic reticulum in muscle fibres. Contraction also results from the application of positively charged macromolecules to the bathing solution. This implies sensitivity of the membrane-associated complex (the cortex system) to an electrical current. A second contractile system may reside in the cytoplasm, where calmodulin is present in contracted hair cells. This protein is a calcium-binding control protein for contraction-like events in smooth muscle and non-muscle cells. The unique presence of the cortex system in outer hair cells, and its absence in inner hair cells, indicates a functional significance that relates to a motor function of outer hair cells in hearing.     

Differential distribution of beta- and gamma-actin in guinea-pig cochlear sensory and supporting cells

Sensory and supporting cells of the mammalian organ of Corti have cytoskeletons containing beta- and gamma-actin isoforms which have been described as having differing intracellular distributions in chick cochlear hair cells. Here, we have used post-embedding immunogold labelling for beta- and gamma-actin to investigate semiquantitatively how they are distributed in the guinea-pig cochlea and to compare different frequency locations. Amounts of beta-actin decrease and gamma-actin increase in the order, outer pillar cells, inner pillar cells, Deiters' cells and hair cells. There is also more beta-actin and less gamma-actin in outer pillar cells in higher than lower frequency regions. In hair cells, beta-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially in the rootlets and towards the periphery of their shafts; labelling densities for gamma-actin differ less between these locations and it is the predominant isoform of the hair-cell lateral wall. Alignments of immunogold particles suggest beta-actin and gamma-actin form homomeric filaments. These data confirm differential distribution of these actin isoforms in the mammalian cochlea and reveal systematic differences between sensory and supporting cells. Increased expression of beta-actin in outer pillar cells towards the cochlear base may contribute to the greater stiffness of this region.     

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.     

An electron microscopic study of the otolithic maculae of the lamprey (Entosphenus japonicus).

The sensory epithelium of the otolithic maculae from the inner ear of the lamprey (Entosphenus japonicus) was studied under the scanning and transmission electron microscope. Two different types of sensory hair cells were discerned and each had a patterned distribution over the various maculae. One type of hair cell had 20-30 short stereocilia and one long kinocilium on its surface. The other type exhibited much longer stereocilia which were arranged in graded heights (organ-pipe configuration) from one side of the cell to the other. So-called striated organelles were found in the cytoplasm of both types of sensory cells. These striated organelles exhibited periodic electron-dense and less dense striations of 170 mum extending from the cuticular plate down to the basal portion of the cell. Generally, this organelle was found in profusion in the sensory cells with long hair bundles but rarely found in the other type of cells.     

Immunohistochemical localization of nonerythroid spectrin (fodrin) in the sensory cells of the vestibular end organs of the rat and guinea pig

Indirect immunofluorescence method was used to study presence and localization of nonerythroid spectrin (fodrin) in the vestibular sensory epithelia. Cryosections of the vestibular organs were treated by monoclonal antibody (mAb) reacting with mammalian fodrin. Strong mAb labelling was observed in the cuticular plate of the vestibular hair cells. The stereocilia were nonreactive and supporting cells showed only a weak reaction. Fodrin seems to have similar localization in the inner ear hair cells as actin (except stereocilia) and it appears to be the major component of the membrane skeleton in the inner ear hair cells. Fodrin-associated membrane skeleton may be involved in the hair cell function in several different ways.     

An immunogold investigation of the distribution of calmodulin in the apex of cochlear hair cells

Calmodulin is found in the mechanosensitive stereociliary bundle of hair cells where it plays a role in various calcium-sensitive events associated with mechanoelectrical transduction. In this study, we have investigated the ultrastructural distribution of calmodulin in the apex of guinea-pig cochlear hair cells, using post-embedding immunogold labelling, in order to determine in more detail where calmodulin-dependent processes may be occurring. Labelling was found in the cuticular plate as well as the hair bundle, the rootlets of the stereocilia being more densely labelled than the surrounding filamentous matrix. In the bundle, labelling was found almost exclusively at the periphery rather than over the centre of the actin core of the stereocilia, and was clearly associated with the attachments of the lateral links that connect them to their nearest neighbours. It was also found to be denser towards the tips of stereocilia compared to other stereociliary regions and occurred consistently at either end of the tip link that connects stereocilia of adjacent rows. The contact region between stereocilia that is found just below the tip link was also clearly labelled. These concentrations of labelling in the bundle are likely to indicate sites where calmodulin is associated with calcium/calmodulin-sensitive proteins such as the various myosin isoforms and the plasma membrane ATPase (PMCA2a) that are known to occur there, and possibly with the transduction channels themselves. At least one of the myosin isoforms, myosin 1c, is thought to be associated with slow adaptation, and PMCA2a with control of calcium levels in the bundle. The concentration of calmodulin in the contact region further supports the suggestion that this is a functionally distinct region rather than a simple geometrical association between adjacent stereocilia.     

人Corti‘s器扫描电镜观察

目的 了解正常人Corti’s器的超微结构特征。方法 用扫描电镜观察成年人和胎儿的Corti’s器。结果成人和胎儿Corti’s器基底圈内、外毛细胞排列规则,中圈和顶圈毛细胞排列不规则,内毛细胞以一排,外毛细胞以三排排列。可观察到列外的内、外毛细胞,四排排列和自然缺失的外毛细胞。外毛细胞粗大的静纤毛以及由于固定不及时引起的外毛细胞静纤毛气球样改变。结论 用扫描电镜观察成人和胎儿Corti’s器的精细结构,发现了毛细胞排列的基本规律和一些异常的形态。及时固定是保存Corti’s器结构完好的必要条件。     

Progression of Inner Ear Pathology in Ames Waltzer Mice and the Role of Protocadherin 15 in Hair Cell Development

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 ^av-J and Pcdh15 ^av-2J ) and two presumptive functional null alleles (Pcdh15 ^av-3J and Pcdh15 ^av-Tg ) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0–P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.     

Microprobe analysis of cryofixed, chemically untreated freeze-dried cochlear hair cells

Elemental (energy-dispersive X-ray) microanalysis was performed of cryofixed, chemically untreated freeze-dried cochlear hair cells of 1-month-old CBA/CBA mice. This technique is very difficult to carry out and time consuming, requiring a large number of specimens to be sectioned, quite at random, in order to obtain absolutely perpendicular section planes. Microprobe determination was performed in different parts of outer and inner hair cells. In addition to the normal intracellular elemental content a high calcium concentration was found at the base of all hair cells. Lower calcium levels were evident in the entire cytoplasm, including the cuticular plate and stereocilia. Technically it was feasible to analyze individual stereocilia.     

Acute ultrastructural changes in acoustic trauma: serial-section reconstruction of stereocilia and cuticular plates

Single-unit recordings were made from populations of auditory-nerve fibers in 12 cats before and after acoustic overstimulation. Cats were killed 4 to 16 h after exposure, and the cochleas were analyzed at the light- and electron-microscopic levels. The exposures were designed to create 40 to 60 dB of acute threshold shift. Physiological changes were similar to those seen in cases of permanent threshold shift: tuning curves with elevation of 'tips' and 'tails' were associated with significant decreases in the mean spontaneous discharge rates; tuning curves with elevated tips but hypersensitive tails were associated with clear elevation of the mean spontaneous rates. At the light-microscopic level, none of the ears showed any significant stereociliary pathology. Some of the ears showed no light-microscopic pathology whatsoever, while others showed signs of swelling and vacuolization in both inner and outer hair cell areas in cochlear regions appropriate to the CF regions showing threshold shifts. The presence or absence of these light-microscopic changes was, to some extent, dependent on the nature of the exposure stimulus. At the electron-microscopic level, in addition to apparent swelling of radial afferent terminals, the inner hair cells themselves were swollen. In two cochlear regions (from two ears) which showed acute threshold shifts of 20 to 40 dB, but no light-microscopic changes, serial-section ultrastructural analysis of stereocilia and cuticular plates was performed. In contrast to the situation in ears with permanent threshold shifts [(1986) Hear Res. 26, 65-88], there was no pathology in the intracuticular portion of the stereocilia rootlets. There were, however, significant changes in the lengths of the supracuticular portion of the rootlets. It is suggested that this attenuation of the supracuticular rootlet could decrease the stiffness of the stereocilia tufts and thereby change the tuning properties and sensitivity of the cochlear partition.     

Immunoelectron microscopic localization of nitric oxide synthase III in the guinea pig organ of Corti

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation. Received: 24 September 1997 / Accepted: 26 June 1998     

Sensory hairs and filaments rods in vestibular hair cells of the waltzing guinea pig. Organization and identification of actin

The waltzing guinea pig suffers from hereditary deafness and vestibular disorder. In vestibular organs, hair cells of Type I develop pathologically and will eventually degenerate. They show fusion of sensory hairs, protrusion of the cuticular plate and contain a rod-shaped inclusion body. With fixation techniques designed to preserve proteins it is shown that this rod has a filamentous substructure reminding one of stereocilia. The packing density of the filaments is similar and circular packing patterns are seen within both structures. However, the rod has an irregular cross-section, as opposed to the circular circumference of stereocilia. The filaments in the rod were identified as containing the protein actin (as those in the stereocilia) by decoration with sub-fragment S-1 of myosin. All filaments in the rod have an identical functional polarity, pointing up from the nucleus towards the cuticular plate. This is contrary to that seen in stereocilia, which have filaments pointing down towards the cuticular plate. It is concluded that the rod is not developed by random polymerization of actin but is the result of co-ordinated assembly reminiscent of that which gives rise to stereocilia. The genetic defect appears to be related to mechanisms which determine the site of nucleation and the functional orientation of actin filaments during development.     

Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure

The distribution of tubulin has been investigated in surface preparations of the guinea pig organ of Corti using indirect immunofluorescence microscopy. Two different monoclonal antibodies to tubulin produce similar distinct patterns of labelling in hair cells. Labelling is greater in inner hair cells than outer hair cells. It occurs in rings around the cell apex, and in a meshwork below and channels through, the cuticular plate. In outer hair cells from the apical region of the cochlea, labelling occurs around the location of a basalward protrusion of the cuticular plate. These patterns correlate with the location of microtubules observed using transmission electron microscopy. A large patch of labelling occurs on the strial side of the cell corresponding to the largest channel through the cuticular plate and the kinociliary basal body. Strands of labelling are seen running parallel to the long axis of the cell between the subcuticular and synaptic region. Many more of these strands are seen in the inner hair cell than the outer hair cell and may correspond to tracks of microtubules transporting neurotransmitter vesicles or other organelles. In outer hair cells, intense labelling and many microtubules are seen in the subnuclear region. The possible roles of the different microtubule arrangements are discussed.     

Spatiotemporal development of cochlear innervation and hair cell differentiation in the rat

The apical cytoskeleton of cochlear hair cells is largely comprised of actin microfilaments and actin-associated proteins, of which fodrin is one of the most prominent. We studied the development of this mechanosensory apical portion of cochlear hair cells of the rat by fluorescence microscopy using rhodamine conjugated phalloidin to detect F-actin and an antibody against alpha-fodrin. An antibody against the 160 kDa neurofilament polypeptide was used for tracing nerve fibers. The first sign of differentiation of the mechanosensory region, actin-containing stereocilia, was observed on the 19th gestational day in the inner hair cells of the basal coil. The appearance of expression of cytoskeletal actin in the cochlear hair cells proceeded gradientally from basal to apical coil and from inner to outer hair cells. Corresponding maturation sequences were observed in the development of fodrin immunoreactivity in the cuticular plates, but the first evidence of this reactivity was found one day later than the appearance of stereocilia in the hair cells at the same location. Also the penetration of neurofilament-positive neurites into the sensory epithelium followed the same kind of longitudinal and radial maturation gradients throughout the cochlea. Fibers were revealed beneath the sensory cells shortly before the first appearance of differentiation of their mechanosensory region. The results suggest that ingrowing nerve fibers may influence the timing of the apical cytoskeleton differentiation in cochlear hair cells or that both these processes could be controlled by the same external signals that are gradientally expressed throughout the cochlea.