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. 2009 Jun 24;102(2):1254–1264. doi: 10.1152/jn.00346.2009

FIG. 6.

FIG. 6.

Imaging and analysis of C17-NBD fluorescent steroid analogues. A: hippocampal neurons were stained with a plasma membrane (PM) marker (CellMask Orange) and NBD-tagged steroids (0.5 μM) were applied for 40 s. Washes with saline and then with 500 μM γ-cyclodextrin (γCDX) lasted for 40 s (each wash). Images were acquired with 4-s interval. NBD fluorescence was always higher for C17-NBD-alphaxalone (A, 56″) and wash with saline was sufficient to remove fluorescence (A, 96″). By contrast, C17-NBD-3α5αA was difficult to remove even after 40-s wash with 500 μM γCDX (A, 136″). Merged images use the NBD-steroid images from 56″. B: fluorescence intensities of selected regions were plotted over time. Intensity values are means ± SE (n = 6). The fluorescence in each compartment was normalized to the last point of steroid application, immediately before saline wash, to emphasize time course differences. Start points of steroid applications and washes are indicated. C17-NBD-3α5αA was not removed by saline and surprisingly exhibited a fluorescence increase in the intracellular compartment during wash with saline. The increase was not as evident in perimembrane regions associated with the plasma membrane marker. C–F: quantitative comparisons of C17-NBD analogues in both intracellular compartments and perimembrane regions. Values are means ± SE (n = 6). C: bar diagrams of normalized fluorescence intensities at the end of steroid application. *P < 0.05 (paired t-test). D: bar diagrams of normalized remaining fluorescence intensities after saline wash (before γCDX application). **P < 0.01 (paired t-test). E: bar diagrams of onset times using the time to reach half the intensity value measured at the end of steroid application (t1/2). *P < 0.05 (paired t-test). F: bar diagrams of offset times during wash with 500 μM γCDX using the time to reach half the intensity value measured at the end of γCDX application (t1/2). Note that the t1/2 for intracellular fluorescence elimination is artificially truncated at the limit of the 40-s wash time. ***P < 0.0001 (paired t-test).