Morphology of labeled efferent fibers in the guinea pig cochlea.

Efferent axons to the guinea pig cochlea were labeled by extracellular injections of horseradish peroxidase into the intraganglionic spiral bundle within the spiral ganglion. The terminal fibers formed by these axons were classified according to their patterns of termination within the basal turn of the cochlea. A class of terminal fibers designated "autonomic" forms a highly branched plexus in the osseous spiral lamina but does not enter the organ of Corti. The termination of single autonomics includes blood vessels as well as areas of the osseous spiral lamina not adjacent to blood vessels. Two major classes of efferent axons from the olivocochlear bundle enter the cochlea by way of the vestibulocochlear anastomosis and terminate either in areas near inner hair cells (IHC efferents) or onto outer hair cells (OHC efferents). The IHC efferents have thin axons throughout their course within the cochlea and can be divided into two subclasses. The most numerous subclass of IHC efferents (unidirectional) enters the inner spiral bundle and turns to spiral in only one direction for less than 1 mm and then forms a discrete termination including many en passant and terminal swellings that are within both the inner and tunnel spiral bundles. A less common subclass of IHC efferents (bidirectional) bifurcates upon entry into the inner spiral bundle to send branches both apically and basally. These terminal fibers take spiral courses that are greater than 1 mm in extent, often course in the tunnel spiral bundle for a large portion of the spiral, and form terminals throughout their extended spiral course. None of the IHC efferent fibers send branches to cross the tunnel to innervate the outer hair cells. A second major class of olivocochlear fibers, OHC efferent fibers, forms large boutons on the outer hair cells, and although they sometimes spiral beneath the IHCs for some length, they do not give off terminals to this region. The OHC efferent axons are thick and myelinated as they enter the cochlea, and they branch near the spiral ganglion to form several terminal fibers. Some of these terminal fibers are thin as they travel from the intraganglionic spiral bundle across the osseous spiral lamina to the organ of Corti, whereas others are thick and obviously myelinated as far peripheral as the habenula.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of EDTA on cytokeratin detection in the inner ear.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.

Reduced neuronal size and dendritic length in the medial superior olivary nucleus of albino rabbits.

We have previously demonstrated that circumscribed structural and functional abnormalities exist in the brainstem auditory system of albino cats. Anomalies in the auditory brainstem evoked response of albino cats were correlated with anatomical defects in the medial superior olivary nucleus (MSO) of the same animals. To examine whether a similar syndrome is present in other albino mammals, we studied the MSO of albino and pigmented rabbits using both Nissl-stained and Golgi-impregnated material. Neurons in the MSO of the albinos were significantly smaller (24%) than those in the pigmented rabbits and there was no overlap in the size distributions between the two groups. Neurons in the abducens nucleus of the albinos were also 14% smaller than in the pigmented rabbits, but this difference was not statistically reliable. The broad overlap in the distributions of neuronal size in the abducens nucleus between groups indicated that not all cells in the albino brainstem are significantly smaller than normal. In the Golgi-impregnated material, the mean total dendritic length for the 'marginal' cell type in the MSO was 39% shorter in albinos than in the pigmented animals. The branching density of dendrites was also significantly reduced in the albinos. Mean total dendritic length for cerebellar granule cells was a statistically insignificant 6% longer in the albinos, demonstrating that dendritic structure is not uniformly affected in all regions of the albino brain. The demonstration of similar anatomical differences in albino rabbits and cats indicates that whatever process produces these effects is not species-specific and may be common to the albinos of other mammalian species. The evidence that the amount of cochlear melanin may be related to differences in auditory function further suggests that the differences in the MSO of the albinos may ultimately be related to absence of inner ear pigmentation and not to other gene effects.

Met-enkephalin characterization in the cochlea: high-performance liquid chromatography and immunoelectron microscopy.

The present paper extends and refines previous observations of enkephalin-like immunoreactivity in the guinea-pig cochlea. Firstly, Met-enkephalin was identified and a quantitative evaluation was made by combining high-performance liquid chromatography (HPLC) and a specific radioimmunoassay. Both the antibody specificity and the HPLC purification allowed us to demonstrate the co-existence, in the cochlea, of at least 3 opioid peptides: Met-enkephalin, Leu-enkephalin and Met-enkephalin-Arg6-Phe7. Secondly, a pre-embedding immunoperoxidase technique was used on whole or dissected cochleas. Immunoreactivity was localized in efferent fibers (coming from the brainstem) in the inner spiral bundle, tunnel spiral bundle and intraganglionic spiral bundle. In the inner spiral bundle vesiculated Met-enkephalin-immunoreactive fibers could be seen synapsing with afferent auditory dendrites. It is hypothesized that these Met-enkephalin immunoreactive fibers (belonging to the lateral efferent system) could be responsible for an inhibitory effect upon the gross cochlear action potential.

Vestibular and cochlear efferent neurons in the monkey identified by immunocytochemical methods.

Attempts were made to identify vestibular (VEN) and cochlear (CEN) efferent neurons in the squirrel monkey using retrograde transport of horseradish peroxidase (HRP) and immunocytochemical methods. HRP implants in the ampulla of the lateral semicircular duct retrogradely labeled cells of VEN bilaterally and some cells of CEN. VEN located lateral to the rostral part of the abducens nucleus formed a compact collection of cells, all of which were immunoreactive only to antisera for choline acetyltransferase (ChAT). CEN, identified by immunoreactivity to ChAT were located at the hilus of the lateral superior olive (LSO), along the lateral border of the LSO and sparsely near lateral parts of the ventral trapezoid nucleus (VTN). A small number of cells and fibers near the border of the VTN and lateral to the LSO were immunoreactive for leucine enkephalin (L-ENK). Fibers immunoreactive for L-ENK also were identified in the hilus of the LSO. No cells of the superior olivary complex were immunoreactive for antisera to ChAT, L-ENK, substance P, gamma-aminobutyric acid or glutamic acid decarboxylase. Cells of VEN and CEN can be identified by their immunoreactivity to ChAT, and some cells and fibers of CEN also contain L-ENK.

Synaptophysin in the developing cochlea.

The immunoreactivity to SY38 (anti-synaptophysin antibody) was investigated in rat and guinea-pig cochleas during development. In rat pups SY38 reactivity first appeared in the inner spiral bundle (below inner hair cells) at postnatal day 3. Later on (days 10 and 15) the basal pole of outer hair cells (OHCs) was also reactive. In fetal guinea-pigs, the inner spiral bundle was reactive on day 45 of gestation, while the reactivity occurred below OHCs on day 62 of gestation. A preliminary electron microscopic finding (from a guinea-pig 62 days of gestation) indicated that SY38 immunoreactivity is localized within varicosities of efferent (olivo-cochlear) endings. Synaptophysin is thus present in the cochlea at the level where the two efferent systems terminate. Moreover, the occurrence of SY38 immunoreactivity, first at the ISB then at the OHC levels, is in accordance with the observation that the maturation of lateral efferents precedes that of medial efferents.

Selective localization of alpha-fetoprotein and serum albumin within the sensory ganglia cells of developing chicken.

The presence of alpha-fetoprotein (AFP) and serum albumin (SA) has been described within developing brain cells of mammals and chicken. In order to avoid the influence of the choroid plexuses in the transport, via the cerebrospinal fluid, of these proteins to brain cells, we studied the presence of AFP and SA within chicken sensory ganglion cells during ontogeny using the indirect immunoperoxidase technique. Our results show that before day 9 of egg incubation, both AFP and SA can be detected within the trigeminal and spinal ganglia cells. Henceforth, the labeling for AFP starts decreasing in the spinal ganglia but not in the trigeminal ganglion. The labeling for SA remains constant in both structures. Differences between AFP and SA staining in the spinal ganglia are maximal on day 11 of incubation, when AFP is no longer detected. It is concluded that at this time, the uptake of AFP by spinal ganglia cells is switched-off while SA is still taken up. As the blood concentration of both proteins is similar at this time, the finding reported here gives support to the advanced hypothesis suggesting the presence of specific receptors for AFP and/or SA in embryonic neural cells.

High-performance liquid chromatographic identification of enkephalin-like peptides in the cochlea.

Met-enkephalin was identified in cochleae from guinea pigs by a combined reversed-phase high-performance liquid chromatographic separation and subsequent radioimmunoassay of the chromatographic fractions. A second Met-enkephalin-immunoreactive fraction was found in the cochlea, with a retention time shorter than that of Met-enkephalin. These findings confirm and extend earlier cytochemical observations, and suggest the differential immunocytochemical staining seen under inner and outer hair cells in the cochlea may be related to different peptides in the two efferent systems innervating the cochlea. Different molecular forms of enkephalin-related peptides may serve different functional or metabolic roles in complex efferent or local regulation of peripheral sensory processing.

Distribution and significance of norepinephrine in the lateral cochlear wall of pigmented and albino rats.

Picogram quantities of norepinephrine were found in cochlear regions of pigmented and non-pigmented rats. These regions of the cochlea were the modiolus, organ of Corti--osseous spiral lamina and the lateral cochlear wall. The content of norepinephrine in the modiolus and lateral cochlear wall of the pigmented rat was significantly greater than that in areas of the non-pigmented rat. In contrast, there was no statistical difference between the norepinephrine content of the organ of Corti--osseous spiral lamina region of the pigmented rat and that of the albino rat. Since a major difference between the pigmented and albino rats is the presence of melanin-containing melanocytes in the modiolus and lateral cochlear wall region of the pigmented animals, it is possible that norepinephrine is stored in cochlear melanocytes.

Glycoconjugates of the tectorial membrane.

The type and quantity of carbohydrate present in the tectorial membrane (TM) was analysed using gas-liquid chromatography and lectin staining of TM protein subunits previously separated by electrophoresis. A relatively large amount of carbohydrate was found, and glucose, N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose and N-acetylneuraminic acid were detected. The presence of mannose and the reaction of many of the protein bands with lectins suggest that at least part of the carbohydrate present is in the form of glycoprotein. The reaction of the main protein band with the lectins RCA1 and ConA is consistent with the suggestion [Thalmann et al. (1985) J. Acoust. Soc. Am. Suppl. 1, Vol. 78, S66] that this band is similar to collagen type II. The failure to detect any uronic acid in these experiments indicates that the more common proteoglycans are probably not a major component of the TM (although keratan sulphate might be present).

GABA-like immunoreactivity in the chick basilar papilla and the lagenar macula.

Gamma-aminobutyric acid (GABA)-like immunoreactivity in the chick basilar papilla and lagenar macula was studied by using anti-serum against GABA coupled with glutaraldehyde to bovine serum albumin. GABA-like immunoreactivity was localized selectively in the cytoplasm of both tall and short hair cells in the basilar papilla, and in the hair cell cytoplasm of the lagenar macula. This immunocytochemical finding suggests that GABA may be an afferent neurotransmitter from the hair cell to the primary afferent neuron in the auditory organs of avians and is consistent with previous findings which suggest that GABA may act as an afferent neurotransmitter in the avian vestibular organs as well. However, the distribution pattern of GABA-like immunoreactivity in the afferent system is quite different from the distribution pattern previously described in the mammalian inner ear where GABA-like immunoreactivity is found exclusively within the efferent system.

Elemental analysis of the early postnatal tectorial membrane.

The analytical electron microscope has revealed that the elemental composition of the early postnatal tectorial membrane is principally the same before and after the development of endolymph. The elemental composition of the tectorial membrane does not give absolute information on the permeability of the membrane to ions such as potassium and chlorine from the endolymphatic space. Such a permeability is, however, likely when comparing the relative peak intensities of chlorine and potassium before and after the development of endolymph.

Lectins demonstrate the presence of carbohydrates in the tectorial membrane of mammalian cochlea.

Histological sections of rat and guinea pig cochleas were exposed to lectins to identify the carbohydrates present in the tectorial membranes. N-Acetylglucosamine, galactose, mannose and fucose were found to be present in both rats and guinea pigs, but N-acetylgalactosamine was not detected. In addition, two control experiments were performed. In the first, each lectin was preincubated with its specific inhibitory sugar. In the second, the unfixed tectorial membranes were exposed to lectins. Radioautographic studies confirmed the presence of glucosamine and fucose in the tectorial membrane of 1-day-old rats.

Proenkephalin and prodynorphin related neuropeptides in the cochlea.

Dynorphin B (rimorphin), a proenkephalin B (prodynorphin)-derived peptide, and met-enkephalin-Arg6, Gly7, Leu8 (met-enkephalin octapeptide), a proenkephalin A-derived peptide, were identified in the mammalian cochlea by specific radioimmunoassays. The antisera are directed against unique sequences in the peptides, and this immunoreactivity cannot be ascribed to cross-reaction with any other known opioid peptides. Met-enkephalin octapeptide and dynorphin B can for this reason serve as reliable markers for the proenkephalin A- and proenkephalin B-derived peptides, respectively. Lesion studies in the cochlea indicate that dynorphin B is confined to olivocochlear efferents. It has not been determined if the dynorphin-containing neurons are the same as those known to contain enkephalin-related peptides, or if they may be cholinergic. Different, presumably inhibitory, neurotransmitters or modulators in the olivocochlear fibers create the possibility of separately modulating the effects of inner or outer hair cells on auditory nerve activity, and so becoming able to study their individual actions in audition. The olivocochlear fiber-hair cell-eighth nerve interaction may provide a valuable model for a complex multi-transmitter synaptic junction.

Immunoreactivity to calcitonin gene-related peptide in the superior olivary complex and cochlea of cat and rat.

In both cat and rat, the cells of origin, axons, and terminals of the lateral olivocochlear system exhibit immunoreactivity to antisera to calcitonin gene-related peptide (CGRP). In the cat, immunoreactive neurons in the brainstem are located in the hilus of the lateral superior olivary nucleus and around its margins. In the rat, immunoreactive neurons are located within the lateral superior olivary nucleus proper. In both species, immunoreactivity in the cochlear duct is limited to the region beneath the inner hair cells. Immunoreactive axons in the cochlear nucleus could not be traced to their source but may arise as collaterals of the lateral olivocochlear system. No other components of the brainstem auditory system react to any extent with the antisera.

Immunohistochemical localization of fibronectin in the chinchilla cochlea.

The purpose of this study was to better understand the molecular composition of the cochlea. Fibronectin (FN), a well characterized adhesive glycoprotein, was localized by immunofluorescence microscopy in fresh and fixed cochlear tissues, and in fixed kidney tissue, using a polyclonal, affinity-purified, rabbit, anti-fibronectin antibody and a secondary antibody coupled to FITC. The FN antibody was free from cross-reactivity with other known basement membrane and cell matrix molecules. FN reactivity in the cochlea was most intense in the basilar membrane, latero-basal borders of Boettcher's cells, otic capsule, endothelial basement membranes (particularly those of the stria vascularis), and as a diffuse, fan-shaped network radiating into the spiral ligament. Little FN labelling was present in the epithelial basement membranes. Negative control tissue showed no immunoreactivity; whereas, positive kidney control tissue showed appropriate FN immunoreactivity in the mesangium of the glomerulus. The most significant finding of this study was that FN is a major component of the basilar membrane and its distribution appears to correspond to the amorphous ground substance. FN was not localized in the organ of Corti or at the tips of the hair-cell stereocilia.

Quantitative evidence for cochlear, non-neuronal norepinephrine.

Endogenous norepinephrine was quantitatively measured in cochlear tissues of pigmented and nonpigmented animals by high-performance liquid chromatography and electrochemical detection. Epinephrine, dopamine and serotonin were not detected. The cochlear norepinephrine content of the pigmented animals was found to be more than double that present in corresponding albinos. Cochlear norepinephrine was only minimally depleted 48 h after surgical removal of the superior cervical ganglion. 12 h after administration of reserpine (5 mg/kg), cochlear norepinephrine was partially depleted. These results indicate that the norepinephrine located in the cochlea is probably not confined entirely to noradrenergic nerve terminals.

Neuron-specific enolase immunoreactivity in the developing mouse cochlea.

Onset of neuron-specific enolase reactivity was observed on gestation day 17 in spiral ganglion cells of the basal coil, and 2 days later in cochlear inner (IHC) and outer (OHC) hair cells. IHCs and OHCs were similarly reactive up to postnatal day 7, then the reactivity began to decrease in OHCs.

Somatostatin along the auditory pathway.

Several structures associated with the auditory pathway have been examined by radioimmunoassay for their content of somatostatin. Of these, the medial geniculate body had the highest content, followed by the cochlear nucleus, inferior colliculus, auditory cortex and cochlea. Cochlear perilymph had no detectable somatostatin-like immunoreactivity.

Tyrosine hydroxylase immunoreactivity identifies possible catecholaminergic fibers in the organ of Corti.

Antibodies to tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase were used in an immunocytochemical examination of catecholamines in the cochlea. In cryostat sections, tyrosine hydroxylase and dopamine beta-hydroxylase-like immunoreactivities fibers were seen in the modiolus that did not extend to the organ of Corti. These corresponded to blood vessel-associated and non-blood vessel-associated fibers that have been previously described with histofluorescence. In surface preparations, tyrosine hydroxylase-like immunoreactivity was seen in the organ of Corti, in the inner and tunnel spiral bundles, suggesting an efferent component may be catecholaminergic.