Organization of microtubules in cochlear hair cells.

The organization of microtubules in hair cells of the guinea-pig cochlea has been investigated using transmission electron microscopy and correlated with the location of tubulin-associated immunofluorescence in surface preparations of the organ of Corti. Results from both techniques reveal consistent distributions of microtubules in inner and outer hair cells. In the inner hair cells, microtubules are most concentrated in the apex. Reconstruction from serial sections shows three main groups: firstly, in channels through the cuticular plate and in a discontinuous belt around its upper perimeter; secondly, forming a ring inside a rim extending down from the lower perimeter of the plate; and thirdly, in a meshwork underlying the main body of the plate. In the cell body, microtubules line the inner face of the subsurface cistern and extend longitudinally through a tubulo-vesicular track between the apex and base. In outer hair cells, the pattern of microtubules associated with the cuticular plate is similar, although there are fewer present than in inner hair cells. In outer hair cells from the apex of the cochlea, microtubules occur around an infracuticular protrusion of cuticular plate material. In the cell body, many more microtubules occur in the region below the nucleus compared with inner hair cells. The possible functions of microtubules in hair cells are discussed by comparison with those found in other systems. These include morphogenesis and maintenance of cell shape; intracellular transport, e.g., of neurotransmitter vesicles; providing a possible substrate for motility; mechanical support of structures associated with sensory transduction.

Intracellular distribution of actin in cells of the organ of Corti: a structural basis for cell shape and motility.

Immunofluorescence staining and phalloidin labeling have provided localization of actin in the sensory and supporting cells of the inner ear at the light microscopic level. However, with electron microscopy, neither actin nor actin filaments have been found in the outer hair cell body. This paper describes various techniques utilized to preserve and identify cytoplasmic actin at the ultrastructural level. Post-embedding staining of Lowicryl K4M sections, pre-embedding staining of permeabilized cells of the organ of Corti, pre-embedding staining of vibratome sections, and pre-embedding staining of permeabilized dissociated cells documented the presence of actin, but each of these techniques was best suited to localize actin in specific parts of the cell. Cytoplasmic actin was labeled when isolated cells were lightly fixed and membranes were permeabilized with detergent--conditions under which the cell ultrastructure was compromised. Under conditions of optimal fixation, cytoplasmic filaments embedded in the dense granular matrix of the hair cell cytoplasm were observed.

Immunoelectron microscopic localization of neurotransmitters in the cochlea.

This paper presents the works and methods of our respective laboratories using electron microscopic immunocytochemistry to identify and localize cochlear neurotransmitters. Antibodies to various prospective neurotransmitters and associated enzymes have been used to study the ultrastructural localization of several candidates for olivocochlear efferent neurotransmitters previously suggested by light microscopic immunocytochemistry. Antibodies against enkephalins label lateral olivocochlear efferent fibers. Antibodies against choline acetyltransferase (ChAT) (an enzyme marker for acetylcholine) label a major population of both lateral and medial efferent fibers and terminals, whereas antibodies to gamma-aminobutyric acid (GABA) label what might be a small subpopulation of both the lateral and medial efferent systems. The GABA-like immunostained medial efferent fibers are preferentially located in the upper turns of the guinea pig cochlea, particularly the third turn. Immunoelectron microscopy shows that neither GABA nor ChAT immunolabels all medial efferent terminals, regardless of cochlear turn. All the different types of immunolabeled efferent terminals have been observed to make characteristic synaptic contacts; lateral efferent terminals on afferent dendrites and medial efferent terminals on outer hair cells and occasionally on type II afferent dendrites. Other types of contacts involving GABA-like, and sometimes met-enkephalin-like, immunostained fibers are occasionally seen particularly in the upper turns of the cochlea. Immunoelectron microscopic results suggest that both medial and lateral efferent systems might be further subdivided on the basis of differences in neurotransmitters. Future trends of immunocytochemical research on cochlear neurotransmitters are proposed, particularly colocalization studies, which show a complex pattern of coexistence of neurotransmitters in the lateral efferent system.

Quantitative distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea.

Distributions of aspartate aminotransferase and glutaminase activities in the guinea pig cochlea have been examined with use of quantitative microchemical techniques to evaluate their roles in cochlear energy metabolism and neurotransmission. Other enzyme activities analyzed were those of choline acetyltransferase and malate dehydrogenase. It is concluded that aspartate aminotransferase activity appears to be especially concerned with cochlear energy metabolism, while glutaminase activity may function in transmitter metabolism in the guinea pig cochlea. Neither enzyme shows a clear association with the olivocochlear bundle.

Identification of precipitable Ca2+ by electron spectroscopic imaging and electron energy loss spectroscopy in the organ of Corti of the guinea pig.

Ca2+ cations were precipitated with potassium antimonate in the cochlea of the guinea pig, and the formed precipitates were localized by electron microscopy using either elastically or inelastically scattered electrons. The elemental composition of the precipitates was determined by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). It was found that calcium, antimony and oxygen were the dominating elements in the precipitates formed in different cell types in the inner ear.

Biochemical features of major organ of Corti proteins (OCP-I and OCP-II) including partial amino acid sequence.

Further biochemical and biophysical characterization of two low-molecular-weight, strongly acidic proteins that are present at extremely high levels in the organ of Corti, tentatively named OCP-I and OCP-II, is presented. The two proteins are also present, although at much lower levels, in the vestibular end-organs and a variety of other inner ear tissues; they have not been observed in other systems. OCP-I and II are highly soluble and do not contain appreciable amounts of carbohydrate. The two proteins, originally described in the guinea pig, are compared electrophoretically with the corresponding proteins in several other mammalian species. Preliminary data on the amino acid composition of the two proteins are presented. Moreover, the amino-terminal sequence of a 22-residue segment of OCP-II is shown and compared to the sequences of known proteins.

Wheat germ agglutinin and Helix pomatia agglutinin lectin binding on cochlear hair cells.

The fluorescein labelled lectins FITC-WGA and FITC-HPA were used to identify specific carbohydrates in cochlear hair cells. Wheat germ agglutinin (WGA) bound with the cell coat of both inner and outer hair cells (IHC and OHC) suggesting the presence of either N-acetyl-D-glucosamine or sialic acid. In contrast, glycoconjugates with terminal N-acetyl-D-galactosamine residues that bind with Helix pomatia agglutinin (HPA), were demonstrated inside the plasma membrane of outer hair cells. WGA and HPA lectin binding implies the presence of anionic glycoconjugates that furnish added negative charge on the membranes to which they are fixed. The presence of sialic acid or N-acetyl-D-glucosamine on the extracellular surface of cochlear hair cell plasma membrane is consistent with the normal distribution of these glycoconjugates in the cell coat. The presence of the membrane associated oligosaccharide N-acetyl-D-galactosamine within the outer hair cell is inconsistent with the distribution of glycoproteins in internal membrane systems of other cell types.

Light and electron microscopic appearance of the inner ear in juvenile ceroid lipofuscinosis (CL).

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.

Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear.

In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.

Light- and electron-microscopic study of gamma-aminobutyric-acid-like immunoreactivity in the guinea pig organ of Corti.

With light and electron microscopy, gamma-aminobutyric-acid (GABA)-like immunoreactivity was examined in the guinea pig organ of Corti. In whole-mount preparations, although GABA-like immunoreactivity was present within efferent components in all turns of the cochlea, it was more intense apically. At the ultrastructural level, GABA-like immunoreactivity was clearly restricted to the efferent system, appearing in axons of the inner and tunnel spiral bundles, in axons crossing the tunnel of Corti, and in terminals filled with numerous labeled vesicles synapsing on outer hair cells.

Localization and fine structure of calcitonin gene-related peptide (CGRP)-like immunoreactive nerve fibres in the organ of Corti of guinea pigs by immunohistochemistry.

The localization and fine structure of calcitonin gene-related peptide (CGRP)-like immunoreactive (CGRPI) fibres in the guinea pig cochlea were examined using immunohistochemistry. Numerous CGRPI fibres were located in the inner spiral bundle corresponding to the lateral system of the olivocochlear bundle. Immunoelectron microscopic analysis demonstrated that the CGRPI fibres belonged to the efferent terminals and some of them made synaptic contacts with peripheral neurites of the auditory nerve. These findings suggested that the olivocochlear CGRP neuron system influenced the neural activity of the auditory nerve, which conveys auditory information from the cochlea to the central nervous system.

Serial section method for cytofluorometric determinations of the DNA content of component cells of the human cochlea.

We describe a method for measuring the DNA content of the component cells of the organ of Corti using serial sections of human cochleae obtained at autopsy. Cochleae were fixed in Carnoy's solution and embedded in Acrytron E, a water-miscible methacrylate resin. A procedure was developed to reduce the background fluorescence in methacrylate-embedded sections; the resin was pretreated with ion-exchange resin (Amberlite IRA-410). Experiments showed that pretreatment reduce the background fluorescence practically to zero. Seventy 3 microns-thick serial sections were prepared on fluorescence free glass slides and stained with azocarmin G and acriflavine-Feulgen. After postirradiation using blue excitation light, the amount of Feulgen-DNA present in the target nucleus in each section was determined using a microfluorometer. The amount of DNA in the entire nucleus was determined by adding together the DNA content of the segments of the nucleus. The characteristic appearance of the organ of Corti made it easy to detect these cells; under green excitation light the cells of this organ exhibited red cytoplasmic azocarmin-G fluorescence. Due to the relatively wide internuclear spaces, cytofluorometry fo individual nuclei could be performed without interference from the neighboring cells. Our technique using serial sections allowed us to measure the DNA content of individual cells and obtain histological information about particular cells and their neighboring cells. Several polyploid cells were found among the Hensen's cells in the cochlea, while all other component cells of the organ of Corti were diploid.

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.

Cupulogenesis and glycoconjugates in the labyrinthine ampulla as revealed by WGA-gold labeling.

We studied the distribution of wheat germ agglutinin (WGA)-bindable glycoconjugates in the vestibular ampulla of mongolian gerbils. WGA was conjugated with gold particles and applied to Lowicryl K4M sections of the ampulla. WGA-binding sites were found on the cupula and some of the secretory granules and Golgi apparatuses in the supporting cells of the sensory epithelia. The granules were seen to secrete into the endolymphatic space through reticular membrane. It is likely, therefore, that glycoconjugates are glycosylated at the Golgi apparatus in the supporting cells, stored in the granules, and secreted through the reticular membrane into the endolymphatic space to be used as a component of the cupula. The cell membranes of various cells, connective tissue filaments in the perilymphatic space and the cytoplasm of melanocytes were also labeled with WGA-gold.

GABA-like immunoreactivity in the squirrel monkey organ of Corti.

The distribution of gamma-aminobutyric acid (GABA)-like immunoreactivity in the squirrel monkey organ of Corti was determined using an antiserum against GABA conjugated to bovine serum albumin. Immunoreactive labeling was seen in the region of the inner spiral bundle, the synaptic region below inner hair cells, in terminals contacting the basal part of outer hair cells, and in tunnel spiral fibers. Examples of each of these immunoreactive components could be observed in all cochlear turns. In the region of inner hair cells, immunoreactive labeling took the form of numerous small puncta randomly distributed below the base of the cells. In the region of outer hair cells, large globular immunoreactive structures reminiscent of terminal endings at the subnuclear level were observed. Since similar structures were seen at the base of outer hair cells in other cochleas processed for AChE, we conclude that GABA-like immunoreactivity was contained in efferent terminals which synapse on outer hair cells. These results strengthen previous evidence for the presence of GABA in the olivocochlear system of the mammalian cochlea.

Met-enkephalin-Arg6-Gly7-Leu8 in the organ of Corti: high performance liquid chromatography and immunoelectron microscopy.

The opioid octapeptide Met-enkephalin-Arg6-Gly7-Leu8 (MERGL) was identified and quantified in the guinea pig cochlea using high performance liquid chromatography and a specific radioimmunoassay. The presence of MERGL immunostaining in efferent endings (coming from the brainstem) within the inner spiral bundle and the tunnel spiral bundle was demonstrated using a pre-embedding immunoelectronmicroscopic technique. Axo-dendritic synapses were observed between the MERGL immunostained varicosities and auditory dendrites. It is hypothesized that MERGL could act, together with Met-enkephalin and other pro-enkephalin A-related peptides, as an efferent neuromodulator or neurotransmitter in the organ of Corti.

Several distinct receptor binding enkephalins in olivocochlear fibers and terminals in the organ of Corti.

Biochemical studies centering on the use of reverse-phase high-performance liquid chromatography (HPLC) and radioimmunoassays (RIA) demonstrate the presence in the guinea pig organ of Corti of at least 3 enkephalin-related peptides, two of which are identified as Met- and Leu-enkephalin, respectively. Enkephalins were identified and quantitated by HPLC-RIA in the isolated second turn of the organ of Corti, but were not found in stria vascularis or auditory nerve dissected from the cochlea. Three enkephalin-immunoreactive HPLC fractions inhibited the binding of labeled naloxone to rat brain membranes. All enkephalins identified by the combined HPLC-RIA procedure had an apparent molecular weight similar to that of Met- and leu-enkephalin peptide standards. Immunocytochemistry, performed with the best-characterized Met-enkephalin antiserum used in the RIAs, localized the enkephalin-like immunoreactivity to lateral efferent fibers and terminals under inner hair cells of the organ of Corti. Other antisera raised against Met-enkephalin, not used for RIA, visualized enkephalin-like immunoreactivity in medial efferent fibers under outer hair cells as well. This enkephalin-like immunoreactivity may reflect the presence in the medial efferent system of other structurally similar peptides in addition to those detected biochemically. Efferent fiber lesion, by evulsion of the vestibular nerve close to the vestibulocochlear anastomosis in which the olivocochlear fibers run, eliminated enkephalin-like immunoreactivity and the enkephalin-related peptides identified by HPLC-RIA.

Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells.

Several structural and contractile proteins have been searched for with immunohistochemical methods using antibodies directed against these proteins. Three types of preparations from the guinea pig have been used: isolated stereocilia from the utricle, organ of Corti fragments obtained by cellular dissociation and 0.2-1 micrometer sections obtained by cryoultramicrotomy. The main finding is that different sets of proteins compose the cytoskeleton in supporting cells and the mechanoreceptor structures of the sensory cells. Thus, actin was found in association with fimbrin in the mechanoreceptive region of hair cells, whereas supporting cells, although rich in actin, did not reveal fimbrin. Instead tubulin was seen together with actin in supporting cells which also exhibited prekeratin. Fimbrin appears to function as a protein capable of making bundles and networks from actin filaments. Its exclusive presence in the mechanosensitive region of the sensory cells is possibly related to the function of these cells as mechanoreceptors.

Adenine nucleotides of the organ of Corti under metabolic stress.

The purpose of the reported experiments was to measure the concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) in the organ of Corti in order to arrive at estimates of three commonly used adenylate ratios. Under normal conditions the concentrations of ATP, ADP, and AMP amounted to 15.8, 3.9, and 0.53 mmoles/kg dry weight, respectively. Of the three substances, AMP is the most sensitive indicator of metabolic stress, since ischemia of 65 seconds leads to an increase of 155%. Under normal conditions the adenylate energy charge, the energy status, and the phosphorylation state amounted to 0.83, 4.1, and 2.5 gram wet weight/mumole, respectively. Within 10 minutes of ischemia the energy charge had declined by 26%, the energy status by 50%, and the phosphorylation state by 76%. The apparent equilibrium constant of adenylate kinase of the organ of Corti was found to be 0.55. The potential significance of these data and their relationship to the situation in the stria vascularis are discussed.

The absence of substance P from guinea pig auditory structures.

The following auditory structures were examined for Substance-P-Containing cells and fibres using a monoclonal antibody: organ of Corti, spiral ganglion, auditory nerve, cochlear nucleus. No substance P was detected in any of these structures. Giving due consideration to the sensitivity and specificity of this method of detection, it was concluded that Substance P has no function in the lower auditory system of the guinea pig.