Spatial and dynamic changes in intracellular Ca 2+ measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Abstract

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca 2+ ([Ca 2+] i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K + (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca 2+] i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca 2+-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca 2+] i in response to stimuli in neurones. Such heterogenic changes in [Ca 2+] i would better be studied by CLMS.