Spatially confined diffusion of calcium in dendrites of hippocampal neurons revealed by flash photolysis of caged calcium

Abstract

The extent of diffusion of a locally evoked calcium surge in dendrites of cultured hippocampal neurons was studied by flash photolysis of caged EGTA. Cells were transfected with pDsRed for visualization, preincubated with caged NP-EGTA (AM) and Fluo-4 (AM) at room temperature and imaged in a PASCAL confocal microscope. Pulses of UV laser light within an active sphere of about 1 μm 2 produced a rise of Fluo-4 fluorescence transients in dendrites which peaked at 1 ms and decayed exponentially with a fast (8–10 ms) time constant. A slower decay component was uncovered following incubation with thapsigargin. Lateral diffusion of [Ca 2+]i did not vary significantly among different size dendrites being symmetric and reaching about 3–3.5 μm at a diffusion rate of 0.8 μm/ms on both sides of the photolysis center. Fluo-4 was also replaced by the membrane-bound Fluo-NOMO (AM) or by the ‘heavy’ Calcium Green dextran (CGd) loaded through a patch pipette. Similar rates of diffusion were found in these cases, indicating that the diffusion is not of the dye complexed to calcium but of genuine free calcium ions. Interestingly, presence of a dendritic spine at the focus of photolysis significantly reduced [Ca 2+]i spread while the focal transient remained unaffected. Finally, [Ca 2+]i diffused about twice as far from the photolysis sphere in glass tubes of a similar diameter to that of a dendrite, indicating that intrinsic calcium uptake mechanisms in the dendrite determine the diffusion of calcium away from its original site of rise.