Sp5 2 upright microscope

Cultured striatal neurons were transfected with a plasmid coding for DARPP-32 fused with carboxy terminal of EGFP (2.5 μg per 35-mm diameter dish) or PKA-GFP (2.5 μg) or EGFP (2.5 μg). FRAP and all imaging experiments were carried out at 37 °C at least 24 h later on a confocal SP5 II upright microscope (Leica Microsystems) with a 40X HCX APO (0.80 NA) water objective at the Institut du Fer à Moulin Cell and Tissue Imaging facility. FRAP and photoactivation experiments were performed with the FRAP Wizard software (Leica Microsystems). During acquisition, the temperature of the sample and the objective was maintained at 37 °C in a heated chamber. The pinhole aperture was opened in such a way that the full width at half the maximal intensity was larger than the depth of the cell. Before photobleaching, 30 images were taken at 0.116-s intervals. After photobleaching of a circular region for 500 ms, 500 images were taken at 0.116-s intervals.

Striatal neurons were infected, after 7–10 days in culture, with Sindbis virus encoding EKAR3 or T-EPAC-VV (mTurquoiseΔ-Epac(CD,ΔDEP)-^cp173Venus-Venus) 6–8 h before the imaging experiments. For cAMP photolysis, 20 min before the experiment culture medium was replaced by ‘imaging medium’. DNMB-cAMP (50 μM) or NPEC-dopamine (100 μM) was added to the imaging medium for cAMP or dopamine photolysis. FRET experiments were carried out at 37 °C on a confocal SP5 II upright microscope (Leica Microsystems) with a 40X HCX APO (0.80 NA) water objective, at the Institut du Fer à Moulin Cell and Tissue Imaging facility. FRET biosensors were excited at 456 nm and fluorescence measured simultaneously in cyan fluorescent protein (CFP, 460–515 nm) and yellow fluorescent protein (YFP, 525–600 nm) channels. cAMP was uncaged at a single 1-μm diameter point with a ultraviolet laser (405 nm) for 1 s. Images were analysed with custom software in Matlab (Matworks). In regions of interest the emission ratio was calculated for each pixel (YFP/CFP for AKAR3 and CFP/YFP for T-Epac-VV) and coded in hue in pseudocolour images.