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Surface glycoconjugates of hair cells and supporting cells in the vestibular endorgans of the bullfrog were identified using biotinylated lectins with different carbohydrate specificities. Lectin binding in hair cells was consistent with the presence of glucose and mannose (CON A), galactose (RCA-I), N-acetylglucosamine (WGA), N-acetylgalactosamine (VVA), but not fucose (UEA-I) residues. Hair cells in the bullfrog sacculus, unlike those in the utriculus and semicircular canals, did not strain for N-acetylglucosamine (WGA) or N-acetylgalactosamine (VVA). By contrast, WGA and, to a lesser extent, VVA, differentially stained utricular and semicircular canal hair cells, labeling hair cells located in peripheral, but not central, regions. In mammals, WGA uniformly labeled Type I hair cells while labeling, as in the bullfrog, Type II hair cells only in peripheral regions. These regional variations were retained after enzymatic digestion. We conclude that vestibular hair cells differ in their surface glycoconjugates and that differences in lectin binding patterns can be used to identify hair cell types and to infer the epithelial origin of isolated vestibular hair cells.

The present study examined the immunocytochemical localization of various intermediate filaments (IFs), 68 kDa, 160 kDa and 200 kDa neurofilament protein (NFP), cytokeratin (CK) 1, 8, 10 and 19, vimentin, and glial fibrillary acidic protein (GFAP) in the vestibular end-organs and ganglia of normal and streptomycin-treated guinea pigs. In normal animals, 68 kDa, 160 kDa and 200 kDa NFP were found in afferent nerve fibers and nerve terminals (probably nerve chalices). Fine nerve fibers (probably efferent and/or sympathetic nerve fibers) were also immunoreactive to NFP. In the vestibular ganglia, 68 kDa and 160 kDa NFP were predominantly distributed in larger cells, whereas 200 kDa NFP was also found in some small ganglion cells. Cytokeratin 8 and 19 were located in supporting cells, transitional cells, dark cells of vestibular end-organs, and the epithelial cell lining of the membranous labyrinth. Vimentin was observed in the hair cells distributed in the central region of the end organs, supporting cells, most connective tissue cells, and Schwann cells of the vestibular ganglion. Although GFAP-like immunoreactivity was evident in glial cells of the proximal vestibular nerve, no immunoreactivity was detected in the distal portion of the vestibular nerve, vestibular ganglion, or vestibular end-organs. These highly distinct staining patterns of IFs indicated that they may play different roles in the different cell types, and that they may serve as a specific marker for each cell type. In streptomycin-treated guinea pigs, immunoreactivities for NFP and vimentin (found in the hair cells) decreased after treatment, whereas immunoreactivities for the other IFs were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of glycoconjugates in the endolymphatic sac (ES) and vestibular end organs from the 12th gestational day (GD) in the developing mouse to the 6th day after birth was analyzed using six biotinylated lectins: wheat germ agglutinin (WGA), Abrus precatorius agglutinin (APA), Ulex europaeus agglutinin I (UEA-I), Ricinus communis agglutinin 120 (RCA120), Helix pomatia agglutinin (HPA), concanavalin A (conA). In the 13th and 15th GD sections, the luminal contents in the fetal ES were strongly labelled with lectins, while only a small amount of substance in the ES was labelled with lectins after the 17th GD. In the 13th GD sections, before the cupula and otoconia were formed, the fetal ES started to produce glycoconjugates labelled with WGA, APA, RCA120 and ConA. In the 17th GD sections, the immature cupula and otoconia were strongly labelled with WGA, APA and RCA120. Sugar residues stained by lectins detected in the substance of the fetal ES were the same as those found in the immature cupula and otoconia. HPA only stained the ES epithelium around the 13th and 15th GD. The fetal ES may interact with the formation of the cupula and otoconia, especially in the early stage during evolution and that HPA-reactive glycoconjugates may be related to the intracellular elements of the ES epithelium only during a well-defined phase of development.

In this report we demonstrate the pattern of calcitonin gene-related peptide (CGRP) mRNA and immunoreactivity in the central and peripheral vestibular system of the rat, using a CGRP cRNA probe and a polyclonal CGRP antiserum. We present evidence that somata in all regions of efferent vestibular neurons contain CGRP based on the correspondence between in situ hybridization (mRNA) and immunohistochemistry (mRNA translation product). CGRP immunohistochemistry (CGRPi) and in situ hybridization confirm that CGRPi axons and terminals present in the vestibular neuroepithelium are efferent in origin. Immunoelectron microscopy revealed an extensive innervation of the afferent vestibular pathway by CGRPi terminals that was not limited to the primary afferent chalice, as previously reported by Tanaka et al. (Brain Res 1989;504:31-5). An efferent neuromodulatory role of CGRP can be inferred from the distribution of terminals found on the primary afferent fibers, and type I and type II hair cells. In addition, we present evidence that nicotinic acetylcholine receptor (nAChR) subunit mRNA is expressed by primary afferent cell bodies. On the basis of these data, a hypothetical molecular mechanism of vestibular efferent modulation of the primary afferent pathway is proposed.

Localization of pyroantimonate-precipitable calcium (Ca) was examined in the vestibular organs of the rat and the guinea pig. Precipitates were observed around the otoconia and in the otolithic membranes of the macula sacculi and macula utriculi. Although the deposition was heavier in the rat than in the guinea pig, the distribution of the Ca-pyroantimonate was similar in both species. Most otoconia in the dark cell area had precipitates around and inside the otoconia. Furthermore, there was some precipitation in the cupulae of the semicircular canals in both animals. The presence of Ca in the precipitates was confirmed by X-ray microanalysis and extraction with EGTA. Localization of the Ca-pyroantimonate around the otoconia in these animals agrees with localization of precipitates of endolymphatic crystals in the tree frog, which are known to grow mainly by accretion. Thus, the otoconia of the rat, and probably also of the guinea pig, can be assumed to grow mainly by accretion.

Vestibular hair cells were isolated from the macula utriculi, macula sacculi and crista ampullaris of the guinea pig, using enzymatic and mechanical techniques. The cells could be classified into two types: flask-shaped ones with a neck and rod- or round-shaped ones without a neck. The flask-shaped cells were thought to be type I cells, the rod-shaped cells type II cells, while the round-shaped cells could have been type I or type II cells. The intracellular free calcium ion concentrations [( Ca2+]i) of these cells were determined using the Ca2(+)-sensitive dye Fura-2 and digital imaging miroscopy. In the resting state, [Ca2+]i in the nucleus and cuticular plate was variable in both types of cells. The 150 mM KCl stimulation, which might induce a depolarization, resulted in a temporary increase in [Ca2+]i in both types of cells. In the presence of 1 micron ionomycin, there was an irreversible increase in [Ca2+]i in both types of cells. These observations aid in elucidating vestibular function.

The presence of carbohydrates in the glycocalyx of the vestibular end organs of the guinea pig was investigated at the ultrastructural level using lectin-gold solutions. The glycocalyx of both the sensory and the supporting cells has variable sugar components. The glycocalyx of the sensory cells including the ciliary interconnections contains N-acetyl-glucosamine, galactose and mannose. In contrast, the glycocalyx of the supporting cells had a lower amount of N-acetylglucosamine. The ciliary interconnections, which have been considered to be a part of the glycocalyx, have larger amounts of galactose and mannose than the surface glycocalyx. These findings indicate that the sugar components may be closely related to the functional significance of the inner-ear glycocalyx and that the functional properties of the glycocalyx may differ between the sensory and the supporting cells of the vestibular end organs.

Vibratome sections were stained for cytochemical localization of carbonic anhydrase (CA) activity in vestibular neuro-epithelium, spiral ligament and spiral limbus. The new finding is the localization of reaction products in the interdental cells of the spiral limbus, Claudius' cells, mesothelial cells of the lower border of spiral ligament, vestibular sensory cells and perilymphatic cells, which have not earlier been proved to have CA activity. The interdental cells showed the products only on the basolateral infoldings. Claudius' cells showed prominent products in the microvilli. In the vestibular sensory cells, the products were present only in the stereocilia and cuticular areas. The perilymphatic fibrocytes under the vestibular sensory epithelium, like the fibrocytes of the spiral ligament, revealed diffuse products throughout the whole cell. In the vestibular supporting cells and transitional cells, the reaction products were localized diffusely in the cytosol, but not in the secretory granules. In the long cell projections of the transitional cells, type II fibrocytes at spiral prominence, mesothelial cells at the uppermost region of the spiral ligament and Borghesan's zone, the localization of the reaction products was the same as that of the basolateral infoldings of the vestibular dark cells and marginal cells of stria vascularis shown previously.

The FITC technique using alpha-bungarotoxin visualized the staining pattern of acetylcholine (ACh) receptors in adult human cochlear and vestibular hair cells (HCs) in normal labyrinths and in cochleae with sensorineural hearing loss. Flourescence staining occurred in the cuticular plates of all HCs, indicating that the micromechanics of their suprastructures can act under cholinergic control. Quantitative differences of the fluorescence of ACh receptors occurred between the three rows of outer HCs at the same level in the cochlea and decreasing along a base-to-apex directed gradient. There is strong evidence that the subsurface cisterns are integrated in the efferent nerve system. In the degenerating organ of Corti an uncoupling of the efferent system takes places adjacent to disintegrating HCs, though the staining in the cuticular plates remains until a very late stage in HC disintegration. In vestibular HCs type I, fluorescence is emitted in the supranuclear area of the cytoplasm below the cuticular plate probably indicating an efferent guidance on the afferent nerve transmission directly via the HC itself.

Molecular biology of the vestibular system has been limited by a number of technical difficulties including fixation, decalcification of the temporal bone and the small size of specific structures relative to their surroundings. In situ hybridization histochemistry and immunoelectron microscopy allow the subcellular study of gene expression and gene products, respectively. We developed the methodologies necessary to apply these techniques to the central and peripheral vestibular systems. The central and temporal bone distributions of the neuropeptide calcitonin gene-related peptide (CGRP) mRNA and two genes coding for gap junction proteins, connexin C32 and C43 mRNA, were studied. The cellular distributions of these mRNAs are presented. In addition, examples of pre-embedding and postembedding immunoelectron microscopy are presented demonstrating the usefulness of these techniques in studying the subcellular localization of specific antigens. The ultrastructural innervation of the vestibular periphery by the efferent neuropeptide CGRP and ultrastructural evidence of glycoprotein secretion by the human endolymphatic sac is presented.

S-100 protein and substance P in the inner ear of the rats on decalcified specimens was investigated immunohistochemically. Immunoreactivity of S-100 protein and substance P was found in various parts of inner ear tissues. These results suggest the possibility of immunohistochemical study of decalcified and paraffinized inner ear tissues.

The immunocytochemical distribution and coexistence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the rat vestibular endorgans were investigated. SP-like immunoreactivity was found in nervous elements beneath and around hair cells. CGRP-like immunoreactivity was also abundantly distributed beneath and within the sensory epithelia. In the present study, double-staining immunocytochemistry revealed that three different types of immunoreactivities: SP-positive/CGRP-negative, SP-negative/CGRP-positive, and SP/CGRP-positive immunostaining can be distinguished. SP/CGRP-immunoreactive fibers were localized within as well as beneath the sensory epithelia. These fibers often penetrated the epithelia and nearly reached the surface. The present immunocytochemical evidence suggests that different types of peripheral nervous systems may exist in the vestibular periphery.

We have previously demonstrated the presence of specific receptors for nerve growth factor (NGF) in cochleovestibular ganglia of 72 h (stage 19-20) quail embryos, with a greater density of NGF receptors in the cochlear portion of the ganglion. The present study was conducted to determine the temporal pattern of NGF receptor expression in cochlear and vestibular ganglia throughout development, and was conducted in two species, quail and mouse. As in the quail, specific binding of 125I-NGF was detected in cochleovestibular ganglia of mouse embryos from an embryonic age equivalent to 72 h quail embryos (embryonic day 11, E11), with a similar concentration of 125I-NGF binding in the cochlear portion. Quantitative studies revealed that 125I-NGF binding continued to increase, in both cochlear and vestibular ganglia, for several days of development, and then began to decrease to minimal levels. Maximal levels were achieved at E7 in the quail, and E14 to E16 in the mouse, while minimal levels were reached by E13 in the quail, and E18 in the mouse. The level of 125I-NGF binding in cochlear ganglia was two to three times higher than in vestibular ganglia; a finding corroborated by radioautographic studies. In both quail and mouse, NGF receptors were more heavily concentrated in the ventromedial portion of the cochlear ganglion, adjacent to the cochlear duct; an area containing both support cells and peripheral neuronal processes. In the vestibular ganglion, 125I-NGF binding was more homogeneous, although small areas containing high densities of silver grains were observed. The presence of NGF receptors in cochlear and vestibular ganglia suggests that these ganglia may be responsive to and/or dependent upon NGF during their development.

Post-embedding immunocytochemical techniques were used to assess distribution of gamma-aminobutyric acid (GABA) in the guinea pig cristae ampullaris. GABA-like immunoreactivity (GABA-LIR) was found in the cytoplasm of both type I (HCI) and type II hair cells (HCII), in the afferent calyx (AC) contacting HCI and some myelinated fibers in the subjacent stroma. HCI and its calyceal contacts showed variation in GABA-LIR, suggesting different populations in HCI and AC. These results support a putative afferent neurotransmitter role of GABA in HC and a possible degradation site of GABA in AC.