Figure 6.
Effects of calcium on PS externalization. a, Sequence of images showing changes in Ca2+i in hair cells in response to the application of 1 mm neomycin. From left to right, the panels show the changes in fura-2 ratio (wide-field imaging) at the times indicated during brief exposure (40 s) to 1 mm neomycin. Times after application, and color scaling for ratio changes are indicated. The asterisk indicates a spontaneous Ca2+ event occurring in the greater epithelial ridge (Tritsch et al., 2007). b, Graph showing the change in fura-2 ratio in hair cells and Hensen's cells after application of 1 mm neomycin. Data are the averages from five cells of the recording in a and are representative of at least three different experiments. c, d, Fluorescence (c, d) and Nomarski interference contrast (c′, d′) images of a basal-coil cochlear culture that was incubated in 1 mm neomycin in nominally calcium-free saline (CFS) for 20 min in the presence of Alexa Fluor 488 annexin V (c, c′). After washout of both compounds, Alexa Fluor 488 annexin V was added in normal, calcium-containing saline and the culture was imaged after 10 min (d, d′). Blebs that formed in the absence of calcium, label with annexin V in the presence of calcium. e, f, Fluorescence (e, f) and Nomarski interference contrast (e′, f′) images of a basal-coil cochlear culture incubated in 5 μm ionomycin in the presence of Alexa Fluor 488 annexin V for 5 min. The image in e is focused on the apical surfaces of the outer hair cells (O1, O2, O3, outer hair cells in rows 1, 2, and 3, respectively), and the image in f is focused on the surfaces of the Hensen's cells (H). g–j, Fluorescence images of 2-d-old basal-coil cochlear cultures incubated for 10 min in Alexa Fluor 488 annexin V in saline with either 250 μm ATP (g), an additional 20 mm CaCl2 (h), an additional 20 mm MgCl2 (i), or 1 mm neomycin (j). k–m, Scanning electron micrographs of 2-d-old basal-coil cochlear cultures incubated for 15 min in saline (k), 20 mm CaCl2 (l), or 1 mm neomycin (m). Scale bars: (in j) c–j, 20 μm; (in m) k–m, 5 μm.