Figure 5.

Time course of FM1-43 dye loading. A, A series of images taken at the time points indicated at the three focal levels L₁, L₂, and L₃ referring to apical, cuticular plate, and basal levels, respectively. Time 0 indicates the onset of dye application (3 μm lasting for 5 sec). The frame is an area measuring 90 × 55 μm. The top row, L₁, shows consecutive images of the bundles of stereocilia at the surface of the hair cells in which a transient peak of dye labeling is observed. The middle row, L₂, shows consecutive images taken at a focal plane close to the cuticular plate at the apical pole of the cell. Dye loads into this region rapidly and remains sustained. The bottom row, L₃, shows images taken at the basal pole of the cell. The dye enters the apex aspects of the cell before being visualized at the basal pole. The approximate time is indicated in seconds. B, The change in fluorescence at the three focal levels as a function of time (adjusted for the interval between frame-capture at each level). Frames were taken at the midsection of the abneural segment of the basilar papilla, and this loading pattern is observed at other locations as well. Dye enters hair cells at the bundle level (L₁) first; the dye is subsequently found at the level of the cuticular plate (L₂) and then is seen finally at the level close to the base of the cell (L₃). The change in fluorescence was fitted with an exponential function, and the time constants (τ, in sec) of FM1-43 dye loading at L₁, L₂, and L₃ were 7, 12, and 17. Mean τ values at L₁, L₂, and L₃ were 6.5 ± 1.8, 11.9 ± 2.8, and 18.3 ± 4.6 (n = 9).