REAL-TIME MEASUREMENTS OF CALCIUM DYNAMICS IN NEURONS DEVELOPING IN-SITU WITHIN ZEBRAFISH EMBRYOS

We have developed a non-invasive technique to measure intracellular ca lcium ([Ca2+](i)) in neurons growing within intact embryos of the zebr afish (Danio rerio). A single blastomere was injected with a calcium-s ensitive fluorescent dye (Calcium Green dextran) between the 32-and 12 8-cell stage and the embryo imaged between 16 h and 20 h postfertilisa tion using laser scanning confocal microscopy. Labelled nerve cells fr om embryos preinjected with dye and dissociated at 16 h showed a fluor escence increase (66+/-22%; n=11) in response to depolarisation with K Cl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca 2+](i),changes in vivo. Imaging of dye loaded cells over 30-min period s in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescenc e changes distinguishable from noise. However, a subset of neurons wit hin the ventral spinal cord exhibited spontaneous, repetitive [Ca2+](i ) oscillations which may have a functional significance during neurona l development.