Local stimulation leads to local recruitment (Experiment). (A) Using high-speed calcium imaging (500 frames/s) we applied a low pressure local stimulus to the left side of the hair bundle (left). Local stimulation led to channel opening and calcium influx near the stimulating pipette leading to fluorescence of the calcium dye Fluo-4ff (black and red arrows). Ca2+ images are taken as an average of 25 frames at the end of the mechanical stimulus (M). Fluorescence traces (bottom graphs) are taken from cilia indicated (arrowheads) in the Ca2+ image. Ca2+ signals decrease moving away from the stimulating pipette (red, green, and blue traces) and eventually are undetectable (magenta and orange traces). The lack of signal in the remaining parts of the bundle is not due to damaged mechano-transduction, because when the pipette is moved back and the pressure increased, functional stereocilia can be seen throughout the whole hair bundle (right panels). All stereocilia where the calcium level was followed for the local stimulation show strong Ca2+ signals when the whole bundle is stimulated. (B) To further illustrate the process of recruitment, we stimulated the hair bundle in (A) at one position with a low pressure and high pressure stimulus. In the low-pressure stimulus (left), Ca2+ signals are low, rise slowly, and get smaller moving more distant to the right of the hair bundle (left bottom traces). When stimulating with higher pressure (right), Ca2+ signals increase in other stereocilia, indicating recruitment of more stereocilia with larger stimulation. Recruitment of stereocilia sometimes entails a delayed activation of channels as seen by Ca2+ signals in stereocilia traces in the right bottom. (C) Current traces for the different stimulations: local (black), whole bundle (red), low pressure (green), and higher pressure (blue). M indicates the time of the drive signal for the mechanical stimulus. To see this figure in color, go online.