[The ultrastructure of utricular maculae of mouse].

Objective: Using scanning electron microscope to observe the ultrastructure of utricular maculae of mouse. Methods: Ten young (6 to 8 weeks) and ten old (>12 months) mice were executed, and their utricles were harvested and the specimens were processed, using scanning electron microscope to observe the structures of the utricles from the surface of otoconia layer to the roots of hair cell cilia. Results: Under the scanning electron microscope, several ultrastructures were observed, including otoconia layer, unstructured gelatinous extracellular matrix layer, honeycomb-like gelatinous extracellular matrix layer, inter-cilia otoconia and hair cell cilia associated with these structures. When compared with young mouse, the otoconia surface of aged mouse was smoother, the gelatinous extracellular matrix between the adjacent otoconias was thinner. Conclusions: Using SEM, ultrastructures can be clearly observed from surface otoconia layer to the roots of hair cell cilia. By the analysis of the ultrastructure of utricular maculae, it is helpful for investigation of the pathological mechanisms of vestibular diseases, such as otolith diseases.

Ultrastructural changes in otoconia of osteoporotic rats.

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.

Morphometry of otoliths in chicken macula lagena.

The macula lagena located at the apical end of the cochlea in birds is characterized by the presence of numerous otoliths with unclear sensory functions. These otoliths are reported to be similar to those in the vestibular system but their detailed features in morphology are unknown. In the present study, we examined the number, size and shape of otoliths from the macula lagena in Chinese domestic chickens (Gallus Ling Nan) with a scanning electron microscope for morphometry. For chickens aged 10-15 post-hatch days, the otoliths in each macula lagena were counted to be 16,055 +/- 4038 (mean +/- S.D., n = 4). The average length and width were 12.98 +/- 3.70 microm and 5.10 +/- 1.48 microm (n = 526 otoliths), respectively. The ratio of length to width for the otolith was 2.58 +/- 0.39 (n = 526 otoliths) and remained relatively constant despite their variations in physical size. Almost all the otoliths were in regular shape and appeared like isolated cylinders with smooth facets at each end, but a few of them (0.025% of 64,221 otoliths screened) were found to be in odd shapes, such as T-shape and cross-shape. The results suggest that otoliths in the macula lagena and those in the vestibular system of bird's inner ear have similar physical properties and may play a similar role in sensing gravitational and acceleration signals.

Regeneration of vestibular otolith afferents after ototoxic damage.

Regeneration of receptor cells and subsequent functional recovery after damage in the auditory and vestibular systems of many vertebrates is well known. Spontaneous regeneration of mammalian hair cells does not occur. However, recent approaches provide hope for similar restoration of hearing and balance in humans after loss. Newly regenerated hair cells receive afferent terminal contacts, yet nothing is known about how reinnervation progresses or whether regenerated afferents finally develop normal termination fields. We hypothesized that neural regeneration in the vestibular otolith system would recapitulate the topographic phenotype of afferent innervation so characteristic of normal development. We used an ototoxic agent to produce complete vestibular receptor cell loss and epithelial denervation, and then quantitatively examined afferent regeneration at discrete periods up to 1 year in otolith maculas. Here, we report that bouton, dimorph, and calyx afferents all regenerate slowly at different time epochs, through a progressive temporal sequence. Furthermore, our data suggest that both the hair cells and their innervating afferents transdifferentiate from an early form into more advanced forms during regeneration. Finally, we show that regeneration remarkably recapitulates the topographic organization of afferent macular innervation, comparable with that developed through normative morphogenesis. However, we also show that regenerated terminal morphologies were significantly less complex than normal fibers. Whether these structural fiber changes lead to alterations in afferent responsiveness is unknown. If true, adaptive plasticity in the central neural processing of motion information would be necessitated, because it is known that many vestibular-related behaviors fully recover during regeneration.

Extremely elongated mitochondria in ionocytes of the saccular epithelium of a teleost, Oreochromis mossambicus (Cichlidae).

Unusually large mitochondria are a rather scarce feature in normal biological tissue and string-like giant mitochondria have hitherto not been reported in animals. Investigating the role of inner ear ionocytes for otolith growth, large ionocytes of the saccular epithelium of the cichlid fish Oreochromis mossambicus were analyzed by imaging of thick sections with energy-filtering transmission electron microscopy. We report here that ionocytes do not contain numerous small-sized mitochondria as has been suggested earlier but rather few, extremely elongated megamitochondria. Since the particular mitochondrial structure is important for normal cell function, such megamitochondria possibly reflect a functional advantage in the context of the presumed role of teleostean ionocytes in regulating the composition of the endolymphatic fluid.

No correlation between multilamellar bodies in the inner ear and further organs of mutant (backstroke, bks) and wildtype zebrafish embryos.

The origin of the proteinacious matrix of the inner ear stones (otoliths) of vertebrates has not yet been clarified. Using the backstroke mutant (bks) of the zebrafish Danio rerio, which is characterized by a complete lack of otoliths, we searched for possibly missing or aberrant structural components within the macular epithelia of the inner ears of embryos on the ultrastructural level. Numerous multilamellar bodies (MLBs) were found. The MLBs were, however, not restricted to the inner ears of mutants but were also found in wildtype individuals and in further organs such as brain and liver. MLBs have hitherto never been described from the inner ear of fish and are generally estimated to be rare structures. Their occurrence in fish liver can, however, be induced by using particular chemical substances, which seem to effect adaptive compensatory processes on the cellular level. Such a chemical treatment also affects the ultrastructure of further organelles. Since the occurrence of MLBs in the liver of zebrafish was not accompanied by an alteration of the morphology of other organelles, their occurrence seems not to be due to environmental stress. The findings indicate that the MLBs cannot be correlated with bks-inherent features as well as with missing otolith development/growth. Since the occurrence of MLBs was independent from the developmental stage of a specimen and its overall tissue preservation, it can moreover be excluded that these MLBs merely represent fixation artifacts. Their presence more likely indicates cellular remodelling processes of hitherto unknown significance.

Localization of the two constitutively expressed nitric oxide synthase isoforms (nNOS and eNOS) in the same cell types in the saccule maculae of the frog Rana pipiens by immunoelectron microscopy: evidence for a back-up system?

There is growing evidence for a nitric oxide/cyclic GMP pathway of signal transduction in the vestibular system. Recently, two isoforms of nitric oxide (NO) synthase (nNOS and eNOS) and NO itself have been identified at the light microscopic level in the vestibulocochlear system of mice using specific antibodies and a new fluorescence indicator. In order to acquire more information about signal transduction and tissue modulation in this neuroepithelium at the cellular and subcellular levels, ultrathin sections of London Resin White-embedded saccule maculae of the frog Rana pipiens were incubated with various concentrations of commercially available antibodies to nNOS and eNOS. The immunoreactivity was visualized by a gold-labelled secondary antibody and the amount of the immunoreactions per microm2 was quantified for the different cell types and subcellular regions. Significant eNOS immunoreactivity was identified in the hair bundles, cuticular plates and the rest of the cytoplasm of the hair cells as well as in different subcellular regions of the supporting cells. Gold-labelled anti-nNOS antibodies stained mainly stereovilli and cuticular structures of hair cells and supporting cells, whereas the number of the immunoreactions in the remaining cytoplasm of both cell types was near the background level. The spatial co-localization of the two NOS isotypes in the same cell regions of hair cells and supporting cells was confirmed in double-labelling experiments. The immunocytochemical findings are suggestive of a redundant system in which one NOS isoform can (partially) replace the other. The different subcellular localization of the NOS isoforms may allow for isoform specific regulation of NOS activity by different Ca2+ currents at the subcellular level, underlining the importance of NO-regulated processes in neuroepithelia of the inner ear.

Ultrastructural analysis of [3H]thymidine-labeled cells in the rat utricular macula.

Ototoxic drugs stimulate cell proliferation in adult rat vestibular sensory epithelia, as does the infusion of transforming growth factor alpha (TGFalpha) plus insulin. We sought to determine whether new hair cells can be regenerated by means of a mitotic pathway. Previously, studies have shown that the nuclei of some newly generated cells are located in the lumenal half of the sensory epithelium, suggesting that some may be newly generated sensory hair cells. The aim of this study was to examine the ultrastructural characteristics of newly proliferated cells after TGFalpha stimulation and/or aminoglycoside damage in the utricular sensory epithelium of the adult rat. The cell proliferation marker tritiated-thymidine was infused, with or without TGFalpha plus insulin, into the inner ears of normal or aminoglycoside-damaged rats for 3 or 7 days by means of osmotic pumps. Autoradiographic techniques and light microscopy were used to identify cells synthesizing DNA. Sections with labeled cells were re-embedded, processed for transmission electron microscopy, and the ultrastructural characteristics of the labeled cells were examined. The following five classes of tritiated-thymidine labeled cells were identified in the sensory epithelium: (1) labeled cells with synaptic specializations that appeared to be newly generated hair cells, (2) labeled supporting cells, (3) labeled leukocytes, (4) labeled cells that we have classified as "active cells" in that they are relatively nondescript but contain massive numbers of polyribosomes, and (5) labeled degenerating hair cells. These findings suggest that new hair cells can be generated in situ by means of a mitotic mechanism in the vestibular sensory epithelium of adult mammals.

Extracellular matrices associated with the apical surfaces of sensory epithelia in the inner ear: molecular and structural diversity.

The ultrastructure and molecular composition of the extracellular matrices that are associated with the apical surfaces of the mechanosensory epithelia in the mouse inner ear are compared. A progressive increase in molecular and structural organization is observed, with the cupula being the simplest, the otoconial membrane exhibiting an intermediate degree of complexity, and the tectorial membrane being the most elaborate of the three matrices. These differences may reflect changes that occurred in the acellular membranes of the inner ear as a mammalian hearing organ arose during evolution from a simple equilibrium receptor. A comparison of the molecular composition of the acellular membranes in the chick inner ear suggests the auditory epithelium and the striolar region of the maculae are homologous, indicating the basilar papilla may have evolved from the striolar region of an otolithic organ. A comparison of the tectorial membranes in the chick cochlear duct and the mouse cochlea reveals differences in the structure of the noncollagenous matrix in the two species that may result from differences in the stochiometry of alpha- and beta-tectorin and/or differences in the post-translational modification of alpha-tectorin. This comparison also indicates that the appearance of collagen in the mammalian tectorial membrane may have been a major step in the evolution of an electromechanically tuned vertebrate hearing organ that operates over an extended frequency range.

Complex vestibular macular anatomical relationships need a synthetic approach.

Mammalian vestibular maculae are anatomically organized for complex parallel processing of linear acceleration information. Anatomical findings in rat maculae are provided in order to underscore this complexity, which is little understood functionally. This report emphasizes that a synthetic approach is critical to understanding how maculae function and the kind of information they conduct to the brain.

The inner ear macular sensory epithelia of the Daubenton's bat.

The inner ear macular sensory epithelia of the Daubenton's bat were examined quantitatively to estimate the area and total number of hair cells. Ultrastructural examination of the sensory epithelium reveals two main types of hair cells: the chalice-innervated hair cell and the bouton-innervated hair cell. The existence of an intermediate type, with a nerve ending covering the lateral side of the hair cell, indicates that the chalice-innervated hair cells are derived from bouton-innervated hair cells. Thus, at least a part of the bouton-innervated hair cells forms a transitional stage. A number of immature as well as apoptotic hair cells were observed. It is suggested that a continuous production of new hair cells takes place in mature individuals, probably based on transdifferentiation of supporting cells.

Vesicular bodies in fish maculae are artifacts not contributing to otolith growth.

The presence, morphology and possible origin of vesicle-like bodies (VBs) within the inner ear macular otolithic membrane of developmental stages of cichlid fish Oreochromis mossambicus and neonate (i.e. functionally fully developed except the reproductive organs) swordtail fish Xiphophorus helleri were analyzed by means of transmission and scanning electron microscopy (TEM and SEM, respectively) employing various fixation procedures. Some authors believe that these VBs are involved in the formation of the organic phase of inner ear otoliths (or statoliths in birds and mammals). Decreasing the osmolarity of the fixation medium from a value rather close to that of native fresh water fish tissue (i.e. 250 mOsm and 290--300 mOsm, respectively) to a value of fixatives mostly employed in TEM studies (ca. 190 mOsm), the amount of VBs increased and the components of sensory inner ear tissue increasingly dilated. Whilst a conventional prefixation with aldehydes followed by osmium tetroxide postfixation yielded numerous VBs, only few of them were observed when the tissue was fixed with aldehydes and osmium tetroxide simultaneously. Therefore, the results demonstrate that inner ear sensory epithelia are extremely sensitive to altering fixation media. On this background it must be concluded that VBs are fixative (i.e. glutaraldehyde) induced artificial structures, so-called membrane blisters. Thus, the protein matrix of otoliths (and possibly that of statoliths in higher vertebrates) is rather provided by secretion processes than by the release of vesicles.

Sciaenid inner ears: a study in diversity.

Sciaenid fishes (Family Sciaenidae) could potentially serve as models for understanding the relationship between structure and function in the teleost auditory system, as they show a broad range of variation in not only the structure of the ear but also in the relationship between the ear and swim bladder. In this study, scanning electron microscopy (SEM) was used to investigate inner ear ultrastructure of the Atlantic croaker (Micropogonias undulatus), spotted seatrout (Cynoscion nebulosus), kingfish (Menticirrhus americanus) and spot (Leiostomus xanthurus). These species reflect the diversity of otolith and swim bladder morphology in sciaenids. The distribution of different hair cell bundle types, as well as hair cell orientation patterns on the saccular and lagenar maculae of these fishes were similar to one another. The rostral ends of the saccular sensory epithelia (maculae) were highly expanded in a dorsal-ventral direction in the Atlantic croaker and spotted seatrout as compared to the kingfish and spot. Also, ciliary bundles of the saccular maculae contained more stereocilia in the Atlantic croaker and spotted seatrout as compared with kingfish and spot. The shapes of the lagenar maculae were similar in all four species. In the Atlantic croaker and spotted seatrout lagenar maculae, the number of stereocilia per bundle was greater than those for the kingfish and spot. Given that saccular macula shape and numbers of stereocilia per bundle correlate with swim bladder proximity to the ear in the studied species, it is possible that inner ear ultrastructure could be indicative of auditory ability in fishes.

Establishment of hair bundle polarity and orientation in the developing vestibular system of the mouse.

The morphological development of the vestibular maculae in the mouse was studied in order to identify elements that may determine how hair-bundle polarity is established. Utricles and saccules develop in parallel. Hair-bundles first appear at embryonic day (E) 13.5. They are initially not polarised and have a kinocilium located at the centre of the cell surface surrounded by stereocilia. Polarisation is rapidly established as the kinocilium becomes eccentrically positioned. The orientation of these polarised bundles is initially not random. It varies systematically across the maculae and the general orientation in utricles is the opposite of that in saccules. At E15.5, in both maculae, hair-bundle orientation angles fall into two populations that differ by approximately 180 degrees defining a line of orientation reversal, the position of which varies little during subsequent maturation. Many more immature hair bundles appear at E15.5 suggesting a second wave of hair cell differentiation is initiated. Otoconial membrane is produced simultaneously across the entire width of both maculae, indicating directional growth of the overlying extracellular matrix is unlikely to influence hair-bundle orientation. Growth of both maculae occurs asymmetrically, essentially outwards from the striola, but it is most pronounced after orientation is defined. Microtubules are prominent in hair cells at the earliest stages of their differentiation, but are oriented parallel to the long axis of the cell and, thus, may not have a role in directing hair-bundle polarity. Microfilament assemblies that are aligned parallel to the apical surface and connect to the adherens junctions in supporting cells could provide a "framework" for hair-bundle orientation. The striated rootlets of ciliary centrioles that are aligned parallel to the cell surface with their tips associated with microfilament assemblies at adherens junctions were the only structural asymmetry identified that might influence the development of hair-bundle polarity.

Ultrastructural localization of ChAT-like immunoreactivity in the human vestibular periphery.

Acetylcholine (ACh) has long been considered a neurotransmitter candidate in the efferent vestibular system of mammals. Recently, choline acetyltransferase (ChAT), the synthesizing enzyme for ACh, was immunocytochemically localized in all five end-organs of the rat vestibule (Kong et al. (1994) Hear. Res. 75, 192-200). However, there is little information in the literature concerning the cholinergic innervation in the vestibular periphery of man. In the present study the ultrastructural localization of the ChAT-like immunoreactivity in the human vestibular periphery was investigated in order to reveal the cholinergic innervation in the human vestibular end-organs. A modified method of pre-embedding immunoelectron microscopy was applied. It was found that the ChAT-like immunoreactivity was located in the bouton-type vesiculated nerve terminals in the vestibular neurosensory epithelia of man. These ChAT-like immunostained nerve terminals make synaptic contacts either with afferent chalices surrounding type I vestibular sensory hair cells, or with type II vestibular sensory hair cells. These results show that the ChAT-like immunoreactivity in the human vestibular periphery is confined to the efferent vestibular system. The ChAT-containing efferents innervate both type I hair cells and type II hair cells, making postsynaptic and presynaptic contacts, respectively. This study presents evidence that ACh is a neurotransmitter candidate in the efferent vestibular system of man.

Ultrastructural localization of GABA-like immunoreactivity in the human utricular macula.

In the vertebrate vestibular periphery, gamma-aminobutyric acid (GABA) has long been presumed to be a neurotransmitter candidate. However, experimental reports about the localization and function of GABA in the vestibular systems of vertebrates are contradictory. In addition, there is no information in the literature concerning the localization of GABA in the human vestibular periphery. The present study investigates the ultrastructural localization of GABA-like immunoreactivity in the human utricular macula. A modified pre-embedding immunostaining electron microscopy technique was applied using two different commercially available polyclonal antibodies to GABA. GABA-like immunoreactivity is confined to the vesiculated nerve fibers and terminals of the human vestibular neurosensory epithelia. The GABA-containing nerve terminals make asymmetrical axo-dendritic synapses with the afferent chalices surrounding the type I sensory hair cells. Type I and type II hair cells as well as afferent chalices are devoid of GABA-like immunoreactive staining. The present study demonstrates that GABA exists in the human vestibular periphery, and that GABA is a neurotransmitter candidate of the human efferent vestibular system.

Effect of cisplatin on the basement membrane anionic sites in the ampulla, macula, and stria vascularis of guinea pigs.

Our objective was to compare changes in the basement membrane anionic sites (BMAs) in the ampulla, macula, and stria vascularis following the infusion of cisplatin (CDDP). After CDDP was administered to anesthetized Hartley guinea pigs, the bony labyrinth was immersed in a solution of polyethyleneimine (PEI). The size and distribution of PEI particles associated with BMAs in the stria vascularis and in the dark cell and sensory cell areas of the vestibular labyrinth were determined by electron microscopy. A significant reduction in the number and size of PEI particles was observed on CDDP-treated strial vessels. The number and size of PEI particles on the basement membranes of the vestibular labyrinth did not differ from those in the control. Our findings suggest that the BMAs of the vestibular labyrinth were not significantly affected by the administration of a single dose of CDDP.

Compartmentalized vesicular traffic around the hair cell cuticular plate.

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.

Morphological evidence for local microcircuits in rat vestibular maculae.

Previous studies suggested that intramacular, unmyelinated segments of vestibular afferent nerve fibers and their large afferent endings (calyces) on type I hair cells branch. Many of the branches (processes) contain vesicles and are presynaptic to type II hair cells, other processes, intramacular nerve fibers, and calyces. This study used serial section transmission electron microscopy and three-dimensional reconstruction methods to document the origins and distributions of presynaptic processes of afferents in the medial part of the adult rat utricular macula. The ultrastructural research focused on presynaptic processes whose origin and termination could be observed in a single micrograph. Results showed that calyces had 1) vesiculated, spine-like processes that invaginated type I cells and 2) other, elongate processes that ended on type II cells pre- as well as postsynaptically. Intramacular, unmyelinated segments of afferent nerve fibers gave origin to branches that were presynaptic to type II cells, calyces, calyceal processes, and other nerve fibers in the macula. Synapses with type II cells occurred opposite subsynaptic cisternae (C synapses); all other synapses were asymmetric. Vesicles were pleomorphic but were differentially distributed according to process origin. Small, clear-centered vesicles, approximately 40-60 nm in diameter, predominated in processes originating from afferent nerve fibers and basal parts of calyces. Larger vesicles approximately 70-120 nm in diameter having approximately 40-80 nm electron-opaque cores were dominant in processes originating from the necks of calyces. Results are interpreted to indicate the existence of a complex system of intrinsic feedforward (postsynaptic)-feedback (presynaptic) connections in a network of direct and local microcircuits. The morphological findings support the concept that maculae dynamically preprocess linear acceleratory information before its transmission to the central nervous system.

[Ultrastructure and peculiarities of the otolith lagena in pigeons].

Rosenhall reported the polarization of kinocilium of otolith organs in the avian inner ear by LM. However, the otolith lagena, which is called the third otolith organ, is not well known, especially in terms of the 3-dimensional relationship between each maculae (utricular maculae, saccular maculae, maculae of the otolith lagena), the details of the striola and otoconial layers, and so on. Therefore, the author conducted a study to clarify these points using 20 Columba Domestica pigeons (40 ears), under the rules for animal experiments established by Nihon University School of Medicine. The pigeons were divided into 4 groups, 1. observations of the membranous labyrinth with binocular microscopes, 2. histological examination of serial sections of inner ear, 3. observations of the otolith lagena by SEM, 4. computer-aided 3-dimensional reconstruction of the membranous labyrinth. The following results were obtained. 1. the mean angle between the utricular maculae and maculae of the otolith lagena was 31 degrees (n = 3), the mean angle between the saccular maculae and maculae of the otolith lagena was 45 degrees (n = 3). 2. striola of the otolith lagena demonstrated a C form and the kinocilium exhibited an orientation identical to that of the striola of the outer saccular maculae, 3. the otolithic membrane of the otolith lagena demonstrated a mesh form and the otoconial layer was observed to be thin above the striola. 4. the surface area of the maculae of the otolith lagena was 0.98mm2 (n = 3) and the number of sensory cells was 16,800 (n = 3). The author also considered the functions of the otolith lagena.