Macrophage and microglia-like cells in the avian inner ear

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia-like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia-like cells increase in the sensory epithelium. The increase in macrophage and microglia-like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia-like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium.     

Evaluation and application of ribotyping for epidemiological studies of Actinobacillus pleuropneumoniae in Denmark

Hair cell regeneration is well documented in the inner ear sensory epithelia of lower vertebrates and birds and may occur in the vestibular organs of mammals. By contrast, hair cell loss in the mature mammalian cochlea is considered irreversible. However, recent reports have suggested that an attempt at hair cell regeneration could occur in vivo in aminoglycoside-lesioned cochleas from neonatal rats. After amikacin treatment, atypical cells with apical specialization reminiscent of early differentiating stereocilia are transiently present at the apex of the intoxicated cochleas but fail to differentiate as hair cells in later stages. In the present study, we used electronic microscopy, histochemistry, and confocal microscopy to investigate the cellular rearrangements in the amikacin-lesioned organ of Corti of rat pups. In addition, we used 5-bromo-2'-deoxyuridine immunocytochemistry to determine whether mitotic processes are involved in the formation of the atypical cells. The morphologic and molecular data suggest that atypical cells are not recovering hair cells, but share characteristics of immature hair cells and supporting cells. Proliferative cells were absent from the region occupied by atypical cells, suggesting that the latter did not arise through mitotic processes. Altogether, the present results support the hypothesis that atypical cells arise through direct transformation of some of the supporting cells that reorganize during hair cell degeneration.     

Hair cell recovery in the vestibular sensory epithelia of mature guinea pigs

The progression of recovery of the vestibular sensory epithelia of guinea pigs after gentamicin-induced hair cell injury was assessed quantitatively and qualitatively. Evaluations were made of the number of cells bearing hair bundles by using scanning electron microscopy (SEM) and of identifiable hair cells in thin sections. Both assessment procedures showed that an initial loss of hair cells in utricular maculae is followed by significant recovery in the number of hair cells present. SEM also showed recovery in saccules comparable to that in utricles. During the recovery, progressive maturation of hair bundles, which exhibited features similar to those seen during normal ontogenetic development of hair cells, could be identified. The pattern and extent of hair cell loss and subsequent reappearance revealed by SEM corresponded with that derived from analysis of thin sections. This suggests that repair of nonlethally damaged hair cells is unlikely but, rather, that new hair cells are produced. An apparent decrease in supporting cell numbers was observed coincident with the increase in hair cell numbers. This complements previous morphological observations, which have suggested new hair cells arise from direct, nonmitotic transdifferentiation of supporting cells. The quantitative analyses indicate that more than half of the hair cells that are lost are replaced, but the recovery process does not result in complete restoration of the epithelium. Eight months after the end of drug treatment, the number of hair cells present was still significantly less than normal, and several other abnormalities persisted. There was also no evidence of any hair cell recovery in the organ of Corti. Thus, there appear to be limitations on the capacity for spontaneous replacement of lost hair cells in the mammalian inner ear.     

Differential glycosylation of auditory and vestibular hair bundle proteins revealed by peanut agglutinin

Up to four morphologically distinct types of cross-link are found between the stereocilia in the hair bundles of avian hair cells. These links are involved in mechanotransduction, force transmission across the bundle, and maintenance of hair bundle structure. They appear to be specialisations of the cell coat, but very little is known about their molecular composition. Chick inner ear tissues were therefore screened with a number of different lectins to find markers for specialisations of the hair bundle surface. One lectin, peanut agglutinin (PNA), which recognises the dissacharide Gal beta 1-3GalNAc, was found to be a fairly selective marker for vestibular hair bundles, but it does not stain the stereocilia of auditory hair cells. The staining patterns observed with PNA in the vestibular system closely resemble those seen with a monoclonal antibody (mab) directed against a 275 kD component of the hair cell's apical surface known as the hair-cell antigen (HCA). However, unlike PNA, the mab recognises both vestibular and auditory hair cells. A detailed comparison of the fluorescence staining patterns observed with PNA and the anti-HCA mab indicates that binding sites for both ligands spatially codistribute on the surface of vestibular hair cells. The lectin and the anti-HCA mab binding sites are both sensitive to trypsin treatment, and, with sections of the vestibular system, PNA pretreatment blocks subsequent anti-HCA mab staining. Immunoelectron microscopy of vestibular hair bundles shows that PNA and the anti-HCA mab both label a type of cross-link known as the shaft connector. This link type is present on both auditory and vestibular hair bundles but reacts with PNA only in the vestibular system. The lectin jacalin, which has greater specificity for Gal beta 1-3GalNAc than does PNA, also only labels vestibular and not auditory hair bundles. Although terminal sialic acid residues can block both PNA and jacalin binding, neuraminidase treatment does not unmask cryptic binding sites for these lectins on auditory hair cells but does reveal PNA and jacalin staining at a number of other locations in the inner ear. The results obtained with the lectins PNA and jacalin indicate that either the HCA or other components of the shaft links are differentially glycosylated in the vestibular and auditory epithelia of the bird. The functional significance for such a difference in glycosylation remains to be determined, but auditory and vestibular hair cells operate over different frequency ranges, and variations in glycosylation might confer different micromechanical properties on the hair bundles in these two systems.     

Transforming growth factor alpha with insulin stimulates cell proliferation in vivo in adult rat vestibular sensory epithelium

Hair cells, the sensory receptors of the mammalian inner ear, have long been thought to be produced only during embryogenesis, and postnatal hair cell loss is considered to be irreversible and is associated with permanent hearing and balance deficits. Little is known about the factors that regulate hair cell genesis and differentiation. The mitogenic effects of insulin and transforming growth factor alpha (TGFalpha) were assayed in vivo in normal and drug-damaged rat inner ear. Tritiated thymidine and autoradiographic techniques were used to identify cells synthesizing DNA. Simultaneous infusion of TGFalpha and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium. New supporting cells and putative new hair cells were produced. Infusion of insulin alone or TGFalpha alone failed to stimulate significant DNA synthesis. These results suggest that exogenous growth factors may have utility for therapeutic treatment of hearing and balance disorders in vivo.     

A role for BDNF in early postnatal rat vestibular epithelia maturation: implication of supporting cells

The early development of the inner ear is largely determined by two members of the neurotrophic family: brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3). Little information is available on the role of these neurotrophins during the late stages of vestibular development in the rat which take place during the first postnatal weeks. At this period where terminal synaptogenesis and maturation occur, we have investigated the expression and the activity of BDNF, the most important neurotrophin in the vestibular system. Using different experimental approaches, we show that BDNF is released by vestibular epithelia on postnatal day 3 (P3) and continues to have a trophic effect on vestibular neurones in vitro. Immunocytochemistry coupled to confocal microscopy revealed a remarkable evolution in BDNF localization during later stages of development. Whereas BDNF is present in both supporting cells and hair cells at P3, its distribution gradually changed and is highly compartmentalized within the upper part of supporting cells at P8 and P15. In parallel, we observed the presence of a truncated form of the BDNF receptor in sensory hair cells. These results suggest an original role for supporting cells, which could be involved in the release of BDNF during the late stages of synaptogenesis in mammalian vestibular epithelia. In particular, BDNF could participate to the set up of the calyx, a specific nerve structure surrounding type I vestibular hair cells.     

Homozygous and frequent deletion of proximal 8p sequences in human prostate cancers: identification of a potential tumor suppressor gene site

The mechanisms for hair cell recovery were investigated after intraortic application of 50 microg gentamicin into the perilymphatic space of the superior semicircular canal of the chinchilla. Histologic evaluation of one normal group and four posttreatment groups (7, 14, 28, and 56 days) was made with light and transmission electron microscopic techniques. The numeric changes of hair cells and supporting cells was quantified with the dissector technique. At 7 and 14 days after treatment, no type I hair cells were present, and 85% and 88% of type II hair cells were lost. Supporting cells decreased to 76% at 7 days, but they recovered to 91% at 14 days. Recovery of the epithelia was evident 28 days after treatment; 83% were type II hair cells, and 3% were type I hair cells. The supporting cell number remained close to normal (86%). Between 14 and 28 days after treatment, there was an increase of 1758 of type II hair cells, representing approximately 125 new hair cells per day. At the same time interval the number of supporting cells remained near normal. These results suggest that new hair cells might be the result of supporting cell mitotic division and differentiation.     

Replacement of lateral line sensory organs during tail regeneration in salamanders: identification of progenitor cells and analysis of leukocyte activity

It has been proposed that supporting cells may be the progenitors of regenerated hair cells that contribute to recovery of hearing in birds, but regeneration is difficult to visualize in the ear, because it occurs deep in the skull. Hair cells and supporting cells that are comparable to those in the ear are present in lateral line neuromasts, and in axolotl salamanders these cells are accessible to microscopic observation in vivo. After amputation of a segment of the tail that contains neuromasts, cells from the posteriormost neuromast on the tail stump divide rapidly and form a migratory regenerative placode. The cells of the regenerative placode represent a lineage that eventually produces both hair cells and supporting cells in replacement neuromasts. We sought to identify the progenitors of the regenerative placode by using differential interference contrast microscopy combined with time-lapse video recording in living axolotl salamanders. In response to amputation, the mantle-type supporting cells at the posteroventral edge of the neuromast that is nearest to the wound increased their frequency of cell division, and gave rise to the first cells of the placode. The increase in mitotic activity of mantle-type supporting cells was accompanied by an unexplained decrease in the frequency of divisions in the same neuromast's population of internal supporting cells. The time-lapse records suggested that the changes in the mitotic activity of supporting cells might have been linked to the presence of phagocytic leukocytes in the vicinity of the neuromast that was nearest to the wound. Leukocytes were evenly distributed around control neuromasts, but during regeneration leukocyte activity increased significantly in the vicinity of the posterior half of the posteriormost neuromast. The redistribution of leukocytes occurred early in the regenerative response, but a causal role for the leukocytes has not been conclusively established. It is possible that the leukocytes could contribute to the formation of the regenerative placode at that location by breaking down the glycocalyx that ensheaths the outermost cells of the neuromast, or through the secretion of mitogenic growth factors.     

Motility of the vestibular hair cell of the guinea pig and bull frog

The motile response of the isolated vestibular hair cell induced by a neurotransmitter was studied. After application of both physostigmine and acetylcholine (Ach) as well as glutamic acid, shortening or tilting of the neck of the guinea pig hair cell was observed. These findings suggest that the effect of a neurotransmitter in the neck region as well as the efferent neuron is involved in the motile response. The location of F-action in isolated vestibular hair cells was investigated by using FITC-labeled phalloidin. In freeze-fixed vestibular hair cells, marked labeling was noted in the hair bundle, cuticular plate and throughout the cytoplasm. After application of both physostigmine and Ach, the labeling in the cuticular plate and the cytoplasm became more intense than that in the hair bundle. Alteration of this phalloidin-labeling pattern suggests that actin could play an important role in the self movement of vestibular sensory cells. The shape of the bull frog hair cell also changed after application of Ach. At the same time, spontaneous discharge and the time constant of the posterior semicircular canal nerve activity decreased. These results suggest that an adaptation mechanism induced by change in the cell shape and membrane potential inhibits the activity of the afferent neuron. Furthermore, these active events could be closely related to the active regulation of vestibular hair cell transmission.     

Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites

A search for Saccharomyces cerevisiae proteins that interact with actin in the two-hybrid system and a screen for mutants that affect the bipolar budding pattern identified the same gene, AIP3/BUD6. This gene is not essential for mitotic growth but is necessary for normal morphogenesis. MATa/alpha daughter cells lacking Aip3p place their first buds normally at their distal poles but choose random sites for budding in subsequent cell cycles. This suggests that actin and associated proteins are involved in placing the bipolar positional marker at the division site but not at the distal tip of the daughter cell. In addition, although aip3 mutant cells are not obviously defective in the initial polarization of the cytoskeleton at the time of bud emergence, they appear to lose cytoskeletal polarity as the bud enlarges, resulting in the formation of cells that are larger and rounder than normal. aip3 mutant cells also show inefficient nuclear migration and nuclear division, defects in the organization of the secretory system, and abnormal septation, all defects that presumably reflect the involvement of Aip3p in the organization and/or function of the actin cytoskeleton. The sequence of Aip3p is novel but contains a predicted coiled-coil domain near its C terminus that may mediate the observed homo-oligomerization of the protein. Aip3p shows a distinctive localization pattern that correlates well with its likely sites of action: it appears at the presumptive bud site prior to bud emergence, remains near the tips of small bund, and forms a ring (or pair of rings) in the mother-bud neck that is detectable early in the cell cycle but becomes more prominent prior to cytokinesis. Surprisingly, the localization of Aip3p does not appear to require either polarized actin or the septin proteins of the neck filaments.     

Postnatal testicular secretions partially restore the disturbances in reproductive activity caused by prenatal hormonal manipulation

A new method of local gentamicin administration was tested in the bullfrog inner ear to achieve ototoxic-induced hair cell destruction. Gelfoam pledgets soaked with known amounts of gentamicin were inserted into the perilymphatic cisterna of the bullfrog through a ventral surgical approach. A dose of 1.20 mg gentamicin, consistent with a perilymphatic concentration of 65 microg/ml, resulted in the desired ototoxic-induced hair cell damage, that is, complete hair cell destruction with minimal disruption of other components of the sensory epithelium. This study demonstrates that this is a useful and simple method to investigate the process of vestibular ototoxicity and hair cell regeneration, including aspects of hair cell destruction and repair.     

Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.     

Effect of ultrasound on the contraction of isolated myocardial cells of adult rats

Microtubules and microfilaments are major cytoskeletal elements in mammalian ova and are important modulators of many fertilization and post-fertilization events. In this study, the integrated distribution of microtubules and microfilaments in pig oocytes were examined under a laser scanning confocal microscope, and the requirements of their assembly during in vitro fertilization and parthenogenesis in in vitro matured pig oocytes were determined. After sperm penetration, an aster of microtubules was produced in the spermatozoon, and this microtubule aster filled the whole cytoplasm during pronuclear movement. During pronuclear formation after activation by insemination, microfilaments became concentrated at the male and female pronuclei and, after electrical stimulation, at the female pronucleus. At metaphase of cleavage, microtubules were detected in the spindle and microfilaments were found mainly in the cortex. At anaphase, microtubule asters assembled at each spindle pole. During cleavage, large asters filled each daughter blastomere and a microfilament-rich cleavage furrow was observed. Cytochalasin B, a microfilament inhibitor, inhibited microfilament polymerization but affected neither pronuclear formation nor movement. However, syngamy and cell division were inhibited in eggs treated with cytochalasin B. Treatment with nocodazole after sperm penetration inhibited microtubule assembly and prevented migration leading to pronuclear union and cell division. These results indicate that microtubule and microfilament assembly in pig oocytes are integrated during fertilization and are required for the union of sperm and egg nuclei and for subsequent cell division.     

What do children aged 5 to 11 years old know about the sun and skin cancer? The practical difficulties of international collaborative research when analysis of language is involved

PURPOSE: The purpose of these studies is to develop an in vitro model of corneal endothelial aging and to investigate age-related changes in morphology, mitosis, prostaglandin synthesis and prostaglandin response pathways. METHODS: First-passage rabbit corneal endothelial cells were grown in vitro for up to 30 days after subculture. PGE2 synthesis was measured by radioimmunoassay. EP2 receptors were evaluated, by determination of PGE2 stimulated by flow cytometry and by bromodeoxyuridine (BrdU) incorporation in subconfluent, confluent and injured cultures. RESULTS: Rabbit corneal endothelial cells become less dense and more irregular in shape as they age in culture, thus resembling their in vivo counterparts. PGE2 synthesis and response decrease with culture age. Injury results in enhanced PGE2 synthesis in both younger and older cultures. In younger cultures, injury also results in mitosis of cells at the wound margin, and this response is greatly diminished in older cultures. CONCLUSIONS: The morphologic and mitotic changes seen in rabbit corneal endothelial cultures in vitro resemble those seen as a consequence of aging in humans and rabbits. Prostaglandin synthesis and response pathways are modified as a result of aging and may play a role in the autocrine regulation of wound repair, especially in younger cells.     

Localization of calretinin mRNA in rat and guinea pig inner ear by in situ hybridization using radioactive and non-radioactive probes

The localization of calretinin mRNA was studied in the rat and guinea pig inner ear by in situ hybridization, and compared to the distribution of the protein previously examined by immunocytochemistry. Radioactive and non-radioactive in situ hybridization (ISH) were performed using oligonucleotide probes labelled with 35S or digoxigenin. Radioactive ISH was more sensitive than non-radioactive ISH. In cochlear and vestibular ganglia, calretinin mRNA was localized in subpopulations of neurons with patterns of distribution similar to those shown by immunocytochemistry. By contrast, the observations in the sensory epithelia differed with the two techniques, ISH revealing less positive structures than immunocytochemistry. Rat inner hair cells and guinea pig inner hair cells, Hensen's cells and Deiters cells, which had been described strongly immunoreactive, appeared positive with radioactive but not with non-radioactive ISH. On the other hand, rat vestibular type II hair cells and guinea pig interdental cells of the spiral limbus which were faintly immunoreactive were not positive with both ISH techniques.     

Role of polo kinase and Mid1p in determining the site of cell division in fission yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

Delta-Notch signalling and the patterning of sensory cell differentiation in the zebrafish ear: evidence from the mind bomb mutant

Mechanosensory hair cells in the sensory patches of the vertebrate ear are interspersed among supporting cells, forming a fine-grained pattern of alternating cell types. Analogies with Drosophila mechanosensory bristle development suggest that this pattern could be generated through lateral inhibition mediated by Notch signalling. In the zebrafish ear rudiment, homologues of Notch are widely expressed, while the Delta homologues deltaA, deltaB and deltaD, coding for Notch ligands, are expressed in small numbers of cells in regions where hair cells are soon to differentiate. This suggests that the delta-expressing cells are nascent hair cells, in agreement with findings for Delta1 in the chick. According to the lateral inhibition hypothesis, the nascent hair cells, by expressing Delta protein, would inhibit their neighbours from becoming hair cells, forcing them to be supporting cells instead. The zebrafish mind bomb mutant has abnormalities in the central nervous system, somites, and elsewhere, diagnostic of a failure of Delta-Notch signalling: in the CNS, it shows a neurogenic phenotype accompanied by misregulated delta gene expression. Similar misregulation of delta ; genes is seen in the ear, along with misregulation of a Serrate homologue, serrateB, coding for an alternative Notch ligand. Most dramatically, the sensory patches in the mind bomb ear consist solely of hair cells, which are produced in great excess and prematurely; at 36 hours post fertilization, there are more than ten times as many as normal, while supporting cells are absent. A twofold increase is seen in the number of otic neurons also. The findings are strong evidence that lateral inhibition mediated by Delta-Notch signalling controls the pattern of sensory cell differentiation in the ear.     

Motility of isolated vestibular hair cells induced by potassium

Many studies of the outer hair cells in cochlea have demonstrated active motility. However, very few studies have been done on vestibular hair cells. This study was designed to demonstrate the motile responses of isolated vestibular hair cells of the chick, induced by potassium promoting contraction. Reversible cell shape changes were observed in 4 of 6 type I hair cells and 3 of 5 type II hair cells by applying the contraction solution. The cell shape changes were revealed mainly in the cuticular plate and infracuticular region. It was suggested that contraction in the cuticular plate of the isolated hair cells might be converted into tension which increases the stiffness of the sensory hairs and restricts their motions, based on the results of the present study, and the structure of contractile proteins and hair behaviors reported by previous investigators.     

Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells

Neurofilaments, the neuron-specific intermediate filaments, are composed of three immunochemically distinct subunits: NF-L, NF-M and NF-H that can be either phosphorylated or unphosphorylated. In mammals, the distribution of these subunits has been described in vestibular ganglion neurons, but there are no reports on the presence of neurofilaments in vestibular hair cells. We investigated, by immunocytochemistry, neurofilaments in vestibular hair cells from rat and guinea-pig using antibodies against the three subunits and to dephosphorylated NF-H (clone SMI 32, recognizes also NF-M on immunoblots), on Vibratome sections of the vestibular end-organs and on isolated hair cells. Various immunostaining protocols were used, as appropriate for the method of observation: laser scanning confocal microscopy (immunofluorescence) and transmission electron microscopy (immunoperoxidase, pre-embedding technique). In rat and guinea-pig cristae and utricles, neurofilament immunoreactivity was observed in axons inside and below the sensory epithelia. In guinea-pig, in addition to this staining, intensely immunoreactive annular structures were found in the basal regions of hair cells. These rings were detected with anti-NF-L, -NF-M and -dephosphorylated NF-H/M antibodies, but not with anti-phosphorylation-independent NF-H. Ring-containing hair cells were present in all regions of the sensory epithelia but were more abundant in the peripheral areas. All levels of observation (Vibratome and thin sections, and isolated hair cells) showed that only the guinea-pig type I hair cells contained a neurofilament ring. High-resolution observations showed that the ring was located below the nucleus, often close to smooth endoplasmic reticulum and the cell membrane.