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.

Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrome type 1 in two families.

Waardenburg syndrome is an autosomal dominant disorder characterized by sensorineural deafness and pigmentary disturbances. Previous work has linked the disease to PAX3 on chromosome 2, and several mutations within the highly conserved paired-box and octapeptide motifs, but not the homeobox, have been reported. In this report, we have used the published cDNA sequence to further define the genomic structure of PAX3, using inverse PCR.

A new nonsyndromic X-linked sensorineural hearing impairment linked to Xp21.2.

X-linked deafness is a rare cause of hereditary hearing impairment. We have identified a family with X-linked dominant sensorineural hearing impairment, characterized by incomplete penetrance and variable expressivity in carrier females, that is linked to the Xp21.2, which contains the Duchenne muscular dystrophy (DMD) locus.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: II. The role of microtubules in the organization of the cuticular plate.

The actin matrix of the cuticular plate, which supports the sensory stereocilia bundle, is coupled to the axial cytoskeleton of the hair cell through a well defined microtubule columnar framework. A collection of axial microtubules in a columnar organization penetrate deep into the dense actin matrix of the cuticular plate. Each microtubule displays at the end a 300-500 nm long fuzzy cap that enmeshes with the actin matrix of the cuticular plate.

Cloning of MITF, the human homolog of the mouse microphthalmia gene and assignment to chromosome 3p14.1-p12.3.

The mouse microphthalmia (mi) gene encodes a basic-helix-loop-helix-zipper protein whose mutations may lead to loss of pigmentation in the eye, inner ear and skin, and to reduced eye size and early onset deafness. Mice with mutations at mi serve as models for human pigment disturbances in skin and eye that may be combined with sensorineural deafness. We have now obtained cDNA and genomic clones of the human homolog of mouse mi, identified a restriction fragment length polymorphism in the gene, and mapped the gene by somatic cell hybrid and fluorescence in situ hybridization techniques to a region of human chromosome 3 that shows a disrupted syntenic conservation with the region on mouse chromosome 6 to which mi maps.

G protein Gi2 alpha in the cochlea: cloning and selective occurrence in receptor cells.

A cDNA library was made from the mouse cochlea and screened with a G protein-cDNA like molecule obtained from cochlear tissue by polymerase chain reaction. The nucleotide sequence of a clone, named cochlear Gi2 alpha, had 99.2% identity to mouse macrophage Gi2 alpha.

Construction of a cDNA library from microdissected guinea pig crista ampullaris.

Poly(A) RNA was isolated from microdissected guinea pig crista ampullaris epithelium and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids electroporated into E. coli.

Cochlear disorder associated with melanocyte anomaly in mice with a transgenic insertional mutation.

We have generated eight lines of transgenic mice containing mouse vasopressin-beta-galactosidase fusion constructs. One of these lines, VGA-9, harbors approximately 50 transgene copies at a single chromosomal site. When bred to transgene homozygosity, mice of this line showed a complete loss of skin pigmentation, microphthalmia, and cochlear abnormalities.

Selective amplification and partial sequencing of cDNAs encoding G protein alpha subunits from cochlear tissues.

An approach utilizing the polymerase chain reaction (PCR) was devised to clone members of a family of cDNAs encoding the alpha subunit of G proteins in the cochlea. RNA was extracted from the whole cochlea of the mouse and from the organ of Corti or the lateral wall of the cochlea microdissected from the guinea pig cochlea. The RNA was reverse-transcribed to cDNA which was selectively amplified by PCR using degenerate primers corresponding to two conserved regions of the G protein coding sequence.

Construction of a cDNA library from microdissected guinea pig organ of Corti.

Poly (A) RNA was isolated from microdissected guinea pig organ of Corti and converted into cDNA with RNase H- murine leukemia virus reverse transcriptase. After size fractionation, the cDNA was directionally ligated into the vector pSPORT 1 and the plasmids were transformed into DH10B E. coli via electroporation.

Analysis of muscarinic receptor subtypes in the mouse cochlea by means of the polymerase chain reaction.

Total RNA was extracted with guanidine thiocyanate from the cochleas of 16-day-old CBAJ mice. The mRNA was purified from the total RNA using oligo-dT cellulose, and the mRNA was treated with DNase to degrade genomic DNA. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR), using primers specific for the nucleotide sequences m1-m5, representing subtypes of muscarinic acetylcholine receptors.

Waardenburg syndrome (WS) type I is caused by defects at multiple loci, one of which is near ALPP on chromosome 2: first report of the WS consortium.

Previous studies have localized the gene for Waardenburg syndrome (WS) type I to the distal portion of chromosome 2q, near the ALPP locus. We pooled linkage data obtained from 41 WS type I and 3 WS type II families which were typed for six polymorphic loci on chromosome 2q in order to refine the location of the WS locus (WS1) and evaluate the extent of genetic heterogeneity. In the course of this work, we developed diagnostic criteria for genetic and phenotypic studies.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane.

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otolithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles.

CGRP-like immunoreactivity in the guinea pig organ of Corti: a light and electron microscopy study.

We examined calcitonin-gene related peptide (CGRP)-like immunoreactivity in the guinea pig organ of Corti at both the light and electron microscope level using immunofluorescence and immunoperoxidase techniques. We observed strong CGRP-like immunoreactivity in the inner spiral bundle and tunnel spiral bundle in all turns at both the light and electron microscope level. CGRP immunostaining was localized exclusively in vesiculated efferent fibers.

Lateral olivocochlear neurons contain both enkephalin and dynorphin immunoreactivities: immunocytochemical co-localization studies.

Antibodies to methionine enkephalin and to dynorphin B were used in an immunocytochemical study examining co-containment of enkephalins and dynorphins in olivocochlear neurons in the guinea pig lateral superior olive. Two methods of sequential co-localization were employed: one using primary antibodies from different species, the second using elution of antibodies. Co-localization of enkephalin-like and dynorphin-like immunoreactivities was found in lateral olivocochlear neurons, suggesting co-containment of enkephalins and dynorphins in this projection pathway from the lateral superior olive to the organ of Corti.

Brain-type prostaglandin D synthetase occurs in the rat cochlea.

Prostaglandin D synthetase [(5Z, 13E)-(15S)-9 alpha, 11 alpha-epidioxy-15-hydroxyprosta-5,13-dienoate D-isomerase, EC 5.3.99.

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.

Electrokinetic shape changes of cochlear outer hair cells.

Rapid mechanical changes have been associated with electrical activity in a variety of non-muscle excitable cells. Recently, mechanical changes have been reported in cochlear hair cells. Here we describe electrically evoked mechanical changes in isolated cochlear outer hair cells (OHCs) with characteristics which suggest that direct electrokinetic phenomena are implicated in the response.

GABA visualized by immunocytochemistry in the guinea pig cochlea in axons and endings of efferent neurons.

Antiserum raised against GABA coupled with glutaraldehyde to bovine serum albumin was applied to the guinea pig cochlea. Immunoreactivity was visualized as horseradish peroxidase reaction product in surface preparations of the organ of Corti using immunocytochemical techniques. Bright-field, differential interference contrast and video-enhanced contrast light microscopy were used.

Tonotopic organization in the inferior colliculus of the rat demonstrated with the 2-deoxyglucose method.

We studied the tonotopic organization in the inferior colliculus of the rat with the 2-deoxyglucose method. Isofrequency bands were observed in the central nucleus of the inferior colliculus. In coronal sections, higher sound frequencies elicited bands that were located more ventrally.

Neurotransmitter-related immunocytochemistry of the organ of Corti.

The principles of immunocytochemistry were outlined in 1942 by Coons et al. and in the 1970's immunocytochemistry emerged as a powerful method for identifying structures and tracing pathways in the nervous system. It now plays a fundamental role in the neuroanatomical and histochemical analysis of the central nervous system.

Immunocytochemical localization of choline acetyltransferase-like immunoreactivity in the guinea pig cochlea.

The immunocytochemical localization of the enzyme choline acetyltransferase (ChAT) was examined in the guinea pig organ of Corti to determine if both lateral and medial systems of efferents would show immunoreactive labeling for this specific enzyme marker of cholinergic neurons. Cochleae were also examined after lesion of efferents to determine if ChAT-like immunoreactivity is confined to efferents. ChAT-like immunoreactivity was seen in the inner spiral bundle, tunnel spiral bundle and by the bases of inner hair cells corresponding to the lateral system of efferents.

Localization of dynorphin B-like and alpha-neoendorphin-like immunoreactivities in the guinea pig organ of Corti.

Antiserum to dynorphin B and antiserum to alpha-neoendorphin were used in an immunocytochemical examination of the guinea pig organ of Corti. Immunoreactive staining for these two proenkephalin B (prodynorphin)-derived peptides was seen in the lateral system of olivocochlear efferents in the organ of Corti: the inner spiral bundle, the tunnel spiral bundle and by the bases of inner hair cells. Immunoreactive staining with both antisera was also seen in efferent terminals on outer hair cells at or above the level of the nucleus, which may represent terminals of either the lateral or the medial system.

Neuron-specific enolase-like immunoreactivity in inner hair cells but not outer hair cells in the guinea pig organ of Corti.

Neuron-specific enolase (NSE) has been localized only in neurons and cells with characteristics of neurons. The immunocytochemical localization of NSE was examined in guinea pig cochleae to determine if hair cells, which have some neuronal characteristics, would show NSE-like immunoreactive labeling. NSE-like immunoreactivity was seen in inner hair cells but not in outer hair cells.