Mechanical stimulation and Fura-2 fluorescence in the hair bundle of dissociated hair cells of the chick.

1. Hair cells were dissociated from the inner ear of a chick and were loaded with the esterified form of the Ca2+-sensitive dye Fura-2. Fura-2 fluorescence was monitored by exciting at wavelengths of 340 and 380 nm in order to monitor intensity changes related to the intracellular Ca2+ concentration. The 340 and 380 nm fluorescence images and the ratio images between them (340 nm/380 nm) gave a good representation of the general shape of the hair cell and the hair bundle. 2. Fluorescence intensities were changed following depolarization of the membrane by high-K+ saline. The 340 nm fluorescence increased while that at 380 nm decreased. These fluorescence changes were probably due to changes in the intracellular Ca2+ concentration. 3. Fluorescence ratio images were observed while applying mechanical stimulation to the hair bundle. The intensity ratio was increased in the hair bundle and in the cell body by the depolarizing stimulation of displacing the hair bundle towards the taller stereocilium, while it was decreased by the hyperpolarizing stimulation of displacing the hair bundle towards the shorter stereocilium. 4. Manganese ions do not pass through the Ca2+ channel but carry transduction current and quench the fluorescence of Fura-2. Quenching was most prominent within the hair bundle when mechanical stimulation was applied by a puff of Mn2+ saline. 5. Smaller-amplitude mechanical stimulation was applied to the hair bundle by a sinusoidal stimulation. The fluorescence intensity ratio was increased about the hair bundle's insertion into the cuticular plate. 6. A belt-shaped depression area emerged in the fluorescence ratio image near the hair bundle's insertion into the cuticular plate when Mn2+ saline was puffed from a distant location, so that the displacement of the hair bundle was produced only by the fringe of the Mn2+ flow. 7. The ratio demonstrated a peak near the hair bundle's insertion into the cuticular plate in Mn2+-free saline exactly where a depression emerged in Mn2+ saline in the same hair cell. A sinusoidal stimulation was applied in both experiments. 8. It is concluded that the fluorescence intensity changes in the hair bundle are due to the influx of Ca2+ or Mn2+ ions through the mechano-electrical transduction channel. The intensity ratio changes observed near the hair bundle's insertion into the cuticular plate might indicate that this is the site of the mechano-electrical transduction.