Fig. 2. Translocation of cPLA₂ in living Purkinje cells in response to glutamate. (A) An example of a Purkinje cell co-expressing GFP-cPLA₂ and DsRed. GFP-cPLA₂ showed a uniform cytosolic distribution in unstimulated cells (upper panels, t=0 seconds), whereas a marked translocation of GFP-cPLA₂ to restricted sites was observed after stimulation with 30 µM glutamate (Glu, lower panels, t=20 seconds). DsRed was expressed to visualize the morphology of Purkinje cells simultaneously (inserts). Note that all z-series sections at 2-µm intervals were combined in two-dimensional xy images. Scale bar, 20 µm. Boxed regions corresponding to the dendrites and soma in the merged images were magnified with orthogonal sections viewing axial directions (zx and zy). Scale bars, 5 µm. (B) Time-lapse images of GFP-cPLA₂ fluorescence following the local application of a 30 µM glutamate solution (t=1-5 seconds, indicated by the horizontal bar). Relative fluorescence intensity (F/F₀) is displayed according to a pseudocolor scale. Scale bar: 20 µm. (C) Traces represent the time course of F/F₀ measured in the indicated areas in B. Five small rectangles delimit the distal spiny dendrite (1), the distal spine (2), the dendritic branch point (3), the proximal main dendrite (4) and the soma (5). The peak time of F/F₀ depends on the distance from the soma. Note that fluorescence is reduced gradually by bleaching.