Fig. 4. Functional intercellular communication measured by the gap-FRAP method. Upper four panels: typical computer-generated images of fluorescence distribution in villous trophoblastic cells cultured in the presence of scrambled antisense for 48 hours measured during a gap-FRAP experiment. After a prebleach scan (A), the fluorescent dye was photobleached in some selected cells (polygons 1 and 2) by means of a strong laser illumination. Isolated cells (polygon 4) kept unbleached served as a control for the spontaneous fading of fluorescent emission (B). The evolution of fluorescence intensities was measured starting just after photobleaching for 12 minutes with a scanning period of 2 minutes. After 12 minutes (C), a fluorescence recovery had occured in area 1, whereas the fluorescence intensity remained weak in area 2, indicating that cell 2 is not coupled to neighboring cells. D represents curves of fluorescence evolution in selected cells: fluorescence recovery in cell 1 follows a closely exponential time-course, reflecting the presence of open gap junctional channels. Note the low decrease of fluorescence intensity in the control unbleached cell (4) due to repeated scanning. Lower panel: percentage of coupled cells between villous trophoblastic cells after 48 hours of culture in the presence of scrambled antisense (dark column) or Cx43 antisense (white column). Coupled cells were characterized by an exponential time-course of fluorescence recovery from neighboring cells into a photobleached test cell. Functional communication was measured between cytotrophoblastic cells, between cyto- and syncytiotrophoblasts and between syncytiotrophoblasts. The number of intercellular contacts analyzed is indicated on top of the bars.