Depletion of intracellular calcium stores did not alter calcium signal propagation across the nuclear envelope. A and B, Fluo-4 calcium imaging experiments using hippocampal neurons stimulated with 100 μM of carbachol; carbachol-induced calcium rises (as shown in A) were abolished in hippocampal neurons pre-treated for 30 min with 30 μM cyclopiazonic acid (B). Images were taken every 1.6 sec. Measurements of individual cells (thin grey lines) and their mean (bold black line) are shown. C and D, propagation of photolysis-induced nuclear (C) or cytosolic (D) calcium signals within the compartment and across the NE in hippocampal neurons after CPA-mediated depletion of intracellular calcium stores. Calcium transients were measured at distances from the uncaging spot of 5 μm (C) and 7 μm (D) in the nucleus (C and D, continuous line) and the cytoplasm (C and D, dotted line). In the inserts, the 5 μm2 areas in the nucleus (C) and in the cytosol (D) that were illuminated with UV light for photolysis of NP-EGTA are indicated with white circles; the positioning of the uncaging spot was guided by MitoTracker staining shown in red. Arrows point towards the positions of calcium measurements (filled white squares) at the indicated distances from the uncaging spot. UV exposure (t
= 16.3 msec) occurred as indicated by the dashed lines. Images were taken every 401 msec. E, time series of raw Fluo-4 fluorescence images taken in the experiments shown in C (lower cell on the right) and D (upper cell on the left). The indicated times correspond to the time scale of the graphs (C and D). The 5 μm2 areas in the nucleus and the cytoplasm, respectively, that were illuminated with UV light for photolysis of NP-EGTA are indicated with white circles. Scale bar is 10 μm. F, comparison of the maximum values (F
) of calcium rises in hippocampal neurons with and without CPA-mediated depletion of intracellular calcium stores. F
values were calculated using exponential curve fits for calcium imaging traces measured at distances of 4 μm, 5 μm, 6 μm, 7 μm, and 8 μm from the uncaging spot; F
values obtained for the various distances were pooled. Calcium imaging data were acquired at 2.5 Hz. Bars represent means ± s.d. (3 independent experiments; for each distance from the uncaging spot, 4 to 5 cells were analyzed in every experiment).