Abstract
We optimized Fluo-4 AM loading of chicken cochlea to report hair-bundle Ca2+ signals in populations of hair cells. The bundle Ca2+ 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 Ca2+ entry through transduction channels; mechanically activating transduction channels increased the Fluo-4 signal, while breaking tip links with Ca2+ chelators or blocking Ca2+ 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 Ca2+ decrease, which could serve to stimulate the hair cell’s tip-link regeneration process. Measurement of bundle Ca2+ 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 Ca2+ signaling in the hair cell.
Highlights
Hair cells of the inner ear are specialized sensory cells that rely on intracellular Ca2+ for essential cellular functions
We correlated live-cell and scanning electron microscopy (SEM) imaging to show the relationship between Fluo-4 fluorescence and the physiological state of hair cells; bundles with moderate Fluo-4 signal at rest had ordered stereocilia and intact tip links, bundles with low Fluo-4 signal were damaged, and very brightly labeled cells were dead or dying
Direct mechanical activation of the transduction channel by fluid jet stimulation resulted in transient increases in Fluo-4 signal in the bundle; blocking the transduction channel and breaking tip links both decreased bundle and cell body Fluo-4 fluorescence, compared to untreated cells
Summary
Hair cells of the inner ear are specialized sensory cells that rely on intracellular Ca2+ for essential cellular functions. In addition to the typical roles that Ca2+ plays in other neuronal cell types, such as modulating neurotransmitter release and synaptic transmission [1], Ca2+ influences three components of mechanotransduction. Intracellular Ca2+ may influence the regeneration of broken tip links [5], transmembrane proteins that gate the transduction channel [6]. The transduction channel is Ca2+-permeable [7], providing a local source of Ca2+ in the mechanically sensitive hair bundle that may modulate these processes; in one model, the predicted–but never before measured–decrease in resting bundle Ca2+ after tip links break is the signal that triggers tip-link regeneration [5]. Typical hair-cell Ca2+ imaging experiments use a whole-cell recording electrode to deliver a fluorescent Ca2+ indicator [8,9]
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