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The distribution of immunoglobulins IgG, IgA and IgM in the inner ear tissue from a patient who died of lung bleeding followed after sepsis was studied, and also the normal guinea pig inner ears and the inner ear disorders induced by Kanamycin injection were studied for the distribution of IgG. The temporal bones were fixed in formaldehyde, decalcified in EDTA and embedded in paraffin. The PAP method was used for the demonstration of the immunoglobulins. In both the human inner ear tissue and the normal control inner ear tissue of the guinea pigs deposits of IgG were found in the sensory organs and the endolymphatic sac, however, in the stria vascularis was slight. The severe damaged inner ears induced by Kanamycin the remarkable decreased deposits of IgG were found in the cochlea, but in the endolymphatic sac the remarkable increased deposits of IgG were found. No IgA and IgM were found in the human inner ear tissue.

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We have investigated the possibility that nerve growth factor (NGF) may play a role in the development of the inner ear. Primordia of the inner ear, the otic vesicle (OV) and cochleovestibular ganglion (CVG), were isolated from 72-hr (stage 19-20) quail embryos and examined for the presence of NGF receptors. Quantitative binding studies revealed that both OV and CVG exhibited specific 125I-NGF binding; levels of nonspecific binding were 6 to 26% of total binding. Scatchard analysis yielded a linear plot, indicating the presence of a single class of NGF receptor. The average binding constant (Kd) was 8.0 nM for OV and 8.6 nM for CVG, corresponding to the low affinity (site II) NGF receptor. Examination of light microscopic radioautographs indicated that most of the specific 125I-NGF binding was located in the ventromedial wall of the OV, with little or no binding in the lateral wall and endolymphatic primordia. These studies were corroborated by microdissection of OV, in which 70% of the radioactivity was found to be localized in the medial half of the OV. In CVG, specific 125I-NGF binding was more concentrated in the cochlear portion of the ganglion, with silver grains primarily over areas containing support cells and immature neurons. Quantitative binding studies with isolated cochlear and vestibular ganglia obtained from 144-hr (stage 29-30) quail embryos revealed that the cochlear ganglion exhibited three times more specific 125I-NGF binding than the vestibular ganglion. The presence of NGF receptors on OV and CVG suggests that these structures are responsive to and/or dependent upon NGF. The following paper (J. Represa and P. Bernd, 1989, Dev. Biol. 134) examines the question of whether NGF serves either as a mitogen, a survival factor, or a differentiation factor in this system.

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A secretory activity has recently been attributed to the endolymphatic sac (ES), as a possible way to contribute to the fluid balance of the entire endolymphatic compartment. Previous histochemical studies have indicated the existence of carbohydrate complexes in the secretory product, both neutral and acidic in nature. A thorough analysis of these compounds in the gerbilline ES was carried out using both transmission electron microscopic (TEM) histochemistry, using dialyzed iron and periodic acid--chromic--silver staining techniques, as well as immunoflourescence with fluorescein-labelled lectins (FITC-lectins). N-acetylglucosamine was found to be one of the major carbohydrate components both of the epithelial layer of the ES and of the luminal precipitate. Proofs for a local secretory activity and its intracellular pathway are presented, suggesting to be involved in the regulation of pressure and volume of inner ear fluids.

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By using well-defined monoclonal antibodies, the distribution of synaptophysin and the glycoprotein Egp-34, the first of its kind, was analyzed immunohistochemically in the fetal and the adult human inner ear. In fetal labyrinths, a distinct immunoreactivity for synaptophysin occurred in the apical region of both outer and inner hair cells as well as in nerve terminals adjacent to both cochlear and vestibular hair cells. In adult hair cells, immunoreactivity was found throughout the cytoplasm. Synaptophysin may act as an important calcium binding protein in the sensory transduction of hair cells. A selective expression of Egp-34 was found in the area of marginal and intermediate cell infoldings in the stria vascularis. A similar localization pattern was found for a number of membrane transport enzymes. The glycoprotein Egp-34 is probably of importance for the active mechanisms regulating the homeostasis of endolymph.

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The carbohydrate contents of the guinea pig endolymphatic sac were investigated by the use of lectins. The lumen of the endolymphatic sac was filled with stainable precipitate containing N-acetyl glucosamine, mannose, glucose, galactose and fucose. N-Acetyl galactosamine was also detected but in minute amounts. This composition corresponded to other areas in the inner ear, such as the cupula, the otolithic membrane and the tectorial membrane. The function of these carbohydrates may play an important role in preventing the lumen of the endolymphatic sac from collapsing as well as in regulating transepithelial fluid transport.

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The anatomical distribution of type II collagen in animal ears was studied by immunohistochemical techniques, using defined monoclonal antibodies to type II collagen. Type II collagen was observed in the cartilage plate of the auricle and external auditory meatus, tympanic annulus, lamina propria of tympanic membrane (pars tensa), interossicular joints, stapes footplate, eustachian tube cartilage, enchondral layer and globuli interossei of the otic capsule, Rosenthal canal, cribriform base, osseous spiral lamina, spiral ligament, limbus, tectorial membrane, semicircular canal membrane and subepithelial layer of the ampullary crista, utricular and saccular maculae, and the endolymphatic duct and proximal part of the sac.

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Inner ear cells were studied by histology, histochemistry and electron microscopy in one case of juvenile form of ceroid lipofuscinosis (Batten's disease). It was found that despite the clinically normal range of auditory acuity (nonaudiometric evaluation) there was a storage process of moderate degree with intralysosomal deposition of a lipopigment of variable ultrastructure with two patterns predominating, curvilinear and fingerprint. Storage was demonstrable in slightly variable degree in every cell type including the receptor cells of the organ of Corti and sensory cells of crista ampullaris. The cochlear neurons displayed a so far unique storage with enormous monovacuolar distension of perikarya comparable only to the lymphocytic vacuolization also present in the juvenile form of the disease. The distension of vacuoles was only partly caused by accumulation of the lipopigment mass and actually led to neuronal deterioration. The results shown here offer a new modell for functional-structural relationship and point to the urgent need of further studies of the inner ear in CL and lysosomal storage generally.

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From neurophysiological and biochemical studies it has been suggested that glycine can function as a major inhibitory neurotransmitter in the central nervous system of mammals. In the present study, anti-glycine antiserum was obtained from rabbits immunized with glycine conjugated to rabbit serum albumin via glutaraldehyde and purified by affinity chromatography. The antibody thus obtained was found specific for glycine as determined by an enzyme immunoassay system. The immunocytochemical distribution of glycine in the auditory tract and internal ear was investigated with the antibody. In the central auditory pathway, glycine-like immunoreactivity was mainly located in the ventral and dorsal cochlear nuclei, trapezoid body, lateral lemniscus and inferior colliculus. In the labyrinth, immunoreactivity was detected in the vestibular ganglion and the supporting cells of the crista ampullaris and the organ of Corti, but not in the spiral ganglion. These findings suggest an important role of glycine in the auditory and vestibular pathways.

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Previous immunohistochemical data have shown that the 44-kDal bone phosphoprotein (44K BPP, also called sialoprotein I or oestopontin) recently isolated in our laboratory was synthesized by osteoblasts and osteocytes and was expressed early during differentiation of bone-forming cells. We report here the presence of 44K BPP antigenicity at certain ectopic sites, namely, the proximal-convoluted tubule of the kidney, neurons, sensory and secretory cells in the internal ear. To insure specificity and reproducibility, different immunohistochemical methods were used and affinity-purified antibodies against two separate preparations of pure 44K BPP were tested. In the cells of the proximal-convoluted tubule, 44K BPP immunoreactivity was observed within apical endocytotic vacuoles and within lysosomes. This staining thus correlates with the degradation of the 44K BPP epitope which we previously demonstrated to occur in serum. On the other hand, in the neurons of the acoustic ganglion and the sensory cells of the macula, 44K BPP immunoreactivity was associated with the Golgi apparatus indicating synthesis and secretion by these cells. The finding that the 44K BPP (or a structurally related molecule) is synthesized by neurons and neuroepithelial cells deserves further investigation with respect to a possible embryologic relationship between neuroectodermal cells and the precursors of some bone forming-cells of the skull.

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Otosclerosis has been hypothesized to result from a disorder of the extracellular matrix of the cartilaginous rests present in the adult temporal bone. Matrix relationship to bone formation and remodeling, as well as the fact that the pathogenesis of otosclerosis is expressed by the action of both of these processes, strongly suggests that more knowledge is needed about the process of otic capsule development. In pursuit of this goal, otic complexes were explanted from mouse embryos that ranged in age from 10.5 to 16 days old and were then exposed to 3H-glucosamine (50 microCi/ml) for 6 hours in vitro. Total labeled glycosaminoglycans (GAGs) and labeled hyaluronate content of each age group of otic explants were measured, and the results were compared to a developmental series of the otic regions of whole embryos stained with either toluidine blue or alcian blue. Increases in the synthesis of the total GAGs were observed on embryonic day 11 and for a prolonged period extending from gestation day 13.5 through day 16. The first increase of GAGs occurred at the initiation of metachromasia and positive staining by toluidine blue of the region of aggregated periotic mesenchyme cells that form the otic capsule. The second increase in GAGs was correlated with chondrification of the capsule. Hyaluronate production revealed a different pattern. Synthesis of hyaluronate exhibits peaks at 10.5, 12.5, and for an extended period of from 13.5 to 14.5 days of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)

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To identify the neurotransmitter released by sensory hair cells, as well as to find other substances that might influence neural function of the inner ear, we have prepared extracts from inner ears of fishes (which have large numbers of hair cells), fractionated the extracts, and studied the effects of the fractionated extracts on the discharge rate of afferent fibers innervating hair cells in the lateral line organ of the African clawed frog Xenopus laevis. The extracts contain active substances that do not bind to a cation-exchange resin at neutral pH. Gel-permeation chromatography suggests that at least 2 unidentified excitatory substances are present in the extracts: one of low molecular weight (Mr about 200) and one of high molecular weight (Mr less than or equal to 5000). Some extracts also contain a high-molecular-weight inhibitory substance (Mr greater than 5000). The low-molecular-weight active substance is detected in extracts of inner ear, but not in brain or muscle. The high-molecular-weight excitatory substance is present both in brain and in inner ear.

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Biochemical analysis of inner ear fluids is fraught with numerous problems. After developing accurate techniques to measure inorganic and organic substances in fluid amounts of 0.1 microliter the problem of measuring and handling series of samples arose. It became necessary to develop methods to handle inner ear fluid taken without sophisticated techniques, i.e. to measure, to store and to dispatch inner ear fluid samples. The fluid volume is measured with a "nanocap" technique and is then transferred to cellulose acetate membranes. A 3 mm long glas capillary tube held by a special device is dipped into the inner ear fluid sample. The capillary tube is then placed on a membrane piece that adheres to the tube. The higher capillary forces of the membrane transfer the fluid out of the glass tube into the membrane which is put into a plastic reaction tube and stored. The reproducibility of the method was tested by flame photometric assays of standard sodium solutions and of perilymph from the scala tympani of guinea pigs. The variation coefficient is below 3% for a standard solution and below 5% for perilymph. The method developed to measure and handle inner ear fluids with the aid of "nanocap" and membrane pieces is easier and faster than the conventional techniques.

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The immunoreactivity for intermediate filaments (IF) and F-actin was documented in serially cryosectioned human inner ears aged 14-19 gestational weeks. An individual immunoreactivity for IF was documented for different cell types in both the cochlear and vestibular parts of the labyrinth. All secretory epithelia showed a similar expression of the cytoskeleton. In outer hair cells, immunoreactivity for vimentin was documented, but not for other IF types. The cytoskeletal composition is similar in the tectorial membrane and in supporting cells of the great epithelial ridge. Strong immunoreactivity for F-actin occurs close to the surface of all vestibular organs and Kölliker's organ, but not to the same extent in other epithelia lining the endolymphatic space.

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Certain epithelia of the human inner ear and human endolymphatic sac display somatostatin and/or somatostatin-like immunoreactivity. Histologic sections from 13 human temporal bones and from 15 endolymphatic sacs were studied using the unlabeled antibody peroxidase-antiperoxidase technique. The somatostatin and/or somatostatin-like immunoreactive cells were located exclusively in the covering epithelium of the spiral prominence and in the epithelium of the intermediate and rugosal part of the endolymphatic sac. In the epithelium of the spiral prominence and endolymphatic sac, secretory granules of the same size and appearance as those of intestinal or pancreatic somatostatin-producing cells were demonstrated ultrastructurally. The findings are consistent with a local exocrine, paracrine, and/or endocrine system of the inner ear.

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Melanin-pigments are found in various parts of the inner ear, especially in the neighbourhood of those epithelia that are believed to be involved in the secretion and/or absorption of endolymphatic fluid. Microprobe analysis (LAMMA, X-ray) measurements were performed in different parts of the inner ear in tissues containing melanin. The tissues were shock-frozen, freeze-dried and embedded in Earle's medium (Spurr). The semi-thin sections used for microprobe analysis were cut dry in a conventional ultramicrotome and mounted on copper grids. Experimental manipulation of the endolymph ionic composition (increased Na+) stimulated the migration of melanosomes from the perinuclear region into the dendritic processes and the rearrangement of the dendritic processes in a close vicinity of the presumably transporting epithelia. The intracellular ion measurements showed the affinity of divalent ions for melanin (Mg++, Ca++, Sr++ and Ba++). It is proposed that melanin represents a physiologically important "reservoir" for essential trace elements and by its binding/release may play a key role in the enzymatically controlled processes of ionic pumps.

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The presence of intermediate filaments in the inner ear of the newborn mouse was analyzed with immunofluorescence techniques using antibodies against the five classes of intermediate filaments: cytokeratins, vimentin, desmin, neurofilaments and glial fibrillary acid protein (GFA). Neurofilaments were found in all nerve fibers from the ganglion cell to the hair cell. In the vestibular ganglion two subpopulations of ganglion cells were identified: a minor part staining intensively with neurofilament and the major part of cells lacking this immunofluorescence. Vimentin occurred in a number of supporting structures in the membranous labyrinth, but not in vestibular or cochlear ganglion cells. Cytokeratins, desmin or GFA were not identified in the inner ear.

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