Calcium distribution and mobilization during depolarization in single cochlear hair cells: Imaging Microscopy and Fura-2

Abstract

Intracellular distribution of cytoplasm-free Ca²⁺ concentrations ((Ca²⁺)i) and dynamic changes during stimulation of viable hair cells were studied using digital imaging microscopy and the Ca²⁺-sensitive dye fura-2. (Ca²⁺)i was visualized on pseudo-colour images and three-dimensional computer graphics. In the resting state, the intra-cellular distribution of (Ca²⁺)i in both the outer and inner hair cells was heterogeneous, and the amount of (Ca²⁺)i in most of the peripheral cytoplasm just beneath the plasma membrane was greater than that throughout the entire cytoplasm. Cell depolarization, induced by elevated K⁺, led to an increase in (Ca²⁺)i in the outer hair cells. The increase in (Ca²⁺)i was not observed under conditions of depolarization in Ca²⁺-free medium. These observations are interpreted to mean that the increase in (Ca²⁺)i is induced by depolarization with the result that there is an influx of extracellular Ca²⁺ into the cytoplasm. When Mn²⁺ was applied during depolarization, a fluorescence quenching occurred. By such means the site of Ca²⁺ channels was elucidated.

Key Words:

• cochlea
• single hair cells
• intra-cellular free Ca²⁺
• fura-2
• digital imaging microscopy