Monitoring intracellular calcium ion dynamics in hair cell populations with Fluo-4 AM

We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca(2+) signals in populations of hair cells. The bundle Ca(2+) signal reported the physiological state of the bundle and cell; extruding cells had very high bundle Fluo-4 fluorescence, cells with intact bundles and tip links had intermediate fluorescence, and damaged cells with broken tip links had low fluorescence. Moreover, Fluo-4 fluorescence in the bundle correlated with Ca(2+) entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca(2+) chelators or blocking Ca(2+) entry through transduction channels each caused bundle and cell-body Fluo-4 fluorescence to decrease. These results show that when tip links break, bundle and soma Ca(2+) decrease, which could serve to stimulate the hair cell's tip-link regeneration process. Measurement of bundle Ca(2+) with Fluo-4 AM is therefore a simple method for assessing mechanotransduction in hair cells and permits an increased understanding of the interplay of tip links, transduction channels, and Ca(2+) signaling in the hair cell.