Effects of inhibition of heterocellular coupling between GL15 cells and astrocytes. A, Photomicrographs in co-cultures (preloading method) showing heterocellular coupling without (upper panel) or with addition of the inhibitor CBX. GL15 donor cells pre-labeled with a non-diffusing membrane-bound dye (DiI, red) and loaded with a fluorescent gap junction-permeable dye (calcein, green) were seeded on a monolayer of unlabeled astrocytes. Functional heterocellular GJC is visualized as the transfer of calcein from DiI-labeled donor cells (small arrows) to surrounding recipients cells. Donors cells appear yellow because of the merge of red and green labeling. CBX reduced the number of calcein-containing (green) recipient astrocytes, (×200). B, Histograms representing mean values of a minimum of three independent runs as in A (range 4 – 6; n = 160 to 225 donor cells per group ± SEM). (***, p < 0.001). C and D, histograms of the migration indices of GL15 in brain slices without or with CBX, illustrating decreased migration in the treated group (mean values ± SEM. **, p < 0.01). E, Cumulative migration indices plotted against time in co-cultures where GL15 cell spheroids were plated upon an astrocyte monolayer, without or with CBX; (S - S0) / S0 values were measured every 12 h, for 48 h. Integrated areas under the two curves in arbitrary units were compared, p < 0.0001.