Mitsuru Ohashi

Histochemical localization of the extracellular matrix components in the annular ligament of rat stapediovestibular joint with special reference to fibrillin, 36-kDa microfibril-associated glycoprotein (MAGP-36), and hyaluronic acid

The annular ligament across the stapediovestibular joint connects the stapes footplate and the vestibular window and plays an important role in the sound conductive system of the ear. In this study, we investigated the distribution of extracellular matrix components in the ligament by histochemical methods at light and electron microscopic levels. As results, light microscopic immunohistochemistry of fibrillin and 36-kDa microfibril-associated glycoprotein (MAGP-36) showed intense immunoreactivities in the annular ligament between the stapes footplate and vestibular window. In addition, the histochemical localization of hyaluronic acid by using biotinylated hyaluronic acid-binding protein (HABP) clarifi ed the presence of hyaluronic acid in the annular ligament. At the electron microscopic level, the immunogold labeling of fibrillin showed intense labeling on the periphery of the electron-dense mantle. Furthermore, the labeling of fibrillin was preferentially seen on the fibrous components among the electronlucent amorphous substance. The immunogold labeling of MAGP-36 was seen on the electron-dense mantle and scattered on the electron-lucent amorphous substance. The gold labeling with biotinylated HABP clearly showed a distribution of hyaluronic acid throughout the amorphous space in the ligament. The present results provide a histochemical profile of the annular ligament of the rat stapediovestibular joint that may provide clues to elucidation of pathological changes in the ligaments and conductive hearing loss in humans.

Three-dimensional regular arrangement of the annular ligament of the rat stapediovestibular joint

The stapes footplate articulates with the vestibular window through the annular ligament. This articulation is known as the stapediovestibular joint (SVJ). We investigated the ultrastructure of adult rat SVJ and report here on the characteristic ultrastructure of the corresponding annular ligament. Transmission electron microscopy showed that this annular ligament comprises thick ligament fibers consisting of a peripheral mantle of microfibrils and an electron-lucent central amorphous substance that is regularly arranged in a linear fashion, forming laminated structures parallel to the horizontal plane of the SVJ. Scanning electron microscopy revealed that transverse microfibrils cross the thick ligament fibers, showing a lattice-like structure. The annular ligament was vividly stained with elastica van Giesons stain and the Verhoeffs iron hematoxylin method. Staining of the electron-lucent central amorphous substance of the thick ligament fibers by the tannate-metal salt method revealed an intense electron density. These results indicate that the annular ligament of the SVJ is mainly composed of mature elastic fibers.

Dissociation enzyme effects on the potassium currents of inner hair cells isolated from guinea-pig cochlea

Tetraethylammonium (TEA)-sensitive potassium currents in the cochlear inner hair cells (IHCs) possess the kinetics of fast inactivation. Some enzymes using for IHCs dissociation affect these inactivation kinetics. IHCs were dissociated from guinea-pig cochlea by 1 mg/ml trypsin or 0.25 mg/ml protease VIII, and the properties of the K+ currents were compared using conventional whole-cell voltage-clamp recordings. TEA-sensitive potassium currents showed fast inactivation kinetics in both trypsin-dissociated cells and protease VIII-dissociated cells. The time constant of the inactivation phase in trypsin-treated cells was similar to that in protease VIII-treated cells. However, the rate of inactivation (compared by the ratio between the steady-state current and initial peak current) in protease VIII-treated cells was larger than that in trypsin-treated cells. In protease VIII-dissociated cells, the time constant of recovery from inactivation elucidated by paired-pulse protocol was 3.5 ms. Papain is another enzyme that is sometimes used for dissociating IHCs, so effects of papain were observed. Extracellular papain application (8 unit/ml) demonstrated a slight increase of the outward potassium currents.

Inactivating and non-inactivating potassium currents in isolated inner hair cells from guinea pig cochlea.

Using conventional whole-cell voltage-clamp recordings, we examined the 4-aminopyridine (4-AP)- and tetraethylammonium (TEA)-sensitive K(+) currents in the cochlear inner hair cells (IHCs) of guinea pigs. 4-AP-sensitive currents were activated slowly and sustained the same current level, whereas TEA-sensitive currents were activated rapidly, followed by inactivation. The inactivation time course of TEA-sensitive currents was voltage-dependent, becoming faster at more depolarized levels. The inactivation of TEA-sensitive currents almost recovered within 5 ms. 4-AP- and TEA-sensitive K(+) currents coexisted in the same IHC.

Effect of cyclopiazonic acid on membrane currents in isolated inner hair cells from guinea-pig cochlea

Cyclopiazonic acid (CPA) is a reticulum-like intracellular Ca(2+) store depletory, which raises intracellular Ca(2+) concentration. The effect of CPA on membrane currents in isolated inner hair cells (IHCs) from guinea-pig cochlea was investigated by the patch-clamp technique in the whole-cell configuration. Four out of eight IHCs showed an augmentation of the currents and the other four cells showed an inhibition of the currents by extracellular CPA application. The activation kinetics of outward currents were not changed by CPA. Three out of four IHCs obtained from the basal part of the cochlea demonstrated augmentation, whereas three out of four IHCs from the apical part demonstrated inhibition of the currents. This result suggests that Ca(2+)-activated currents were dominant in the basal IHCs of the cochlea.

Glutamate induced currents in isolated inner hair cells from guinea-pig cochlea

We investigated the direct action of glutamate (Glu) on the membrane current of isolated inner hair cells of guinea-pig cochlea. Glu elicited inward currents at a holding potential of -70 mV. Eight of 13 cells showed a steady inward current, while five of 13 cells showed a fast and rapidly desensitized current. I-V relationships demonstrated that the reversal potential of Glu-induced current was near 0 mV. Glu-induced currents were dose-dependent, where the half maximum concentration (K(d)) was 41 microM and Hill coefficient (n) was 1.75.