Dm6000

Image acquisition was performed using a ×100 objective (NA 1.40) on a Leica (Nussloch, Germany) DM6000 upright epifluorescence microscope with a 12-bit cooled CCD camera (Micromax, Roper Scientific) run by MetaMorph software (Roper Scientific, Evry, France). Quantification was performed using MetaMorph software (Roper Scientific). Image folders were randomized before analysis. For morphological spine analysis exposure time was adjusted for each eGFP image to obtain best fluorescence to noise ratio and to avoid pixel saturation. For each neuron a well-focused dendrite was chosen, spine heads were manually delimited and their area were quantified. To assess KCC2-Flag clusters, exposure time was fixed at a non-saturating level and kept unchanged between cells and conditions. For cluster analysis, images were first flatten background filtered (kernel size, 3 × 3 × 2) to enhance cluster outlines, and a user defined intensity threshold was applied to select clusters and avoid their coalescence. Clusters were outlined and the corresponding regions were transferred onto raw images to determine the mean KCC2–Flag cluster number, area and fluorescence intensity. The dendritic surface area of the region of interest was measured to determine the number of clusters per 10 µm². For each culture, we analyzed ~10 cells per experimental condition and ~100 clusters or ~15 spines per cell.

Image acquisition was performed using a 63× objective (NA 1.32) on a Leica (Nussloch, Germany) DM6000 upright epifluorescence microscope with a 12-bit cooled CCD camera (Micromax, Roper Scientific) run by MetaMorph software (Roper Scientific, Evry, France). Image exposure time was determined on bright cells to obtain best fluorescence to noise ratio and to avoid pixel saturation. All images from a given culture were then acquired with the same exposure time and acquisition parameters. For cluster colocalization analysis, quantification was performed using MetaMorph software (Roper Scientific). For each image, several dendritic regions of interest were manually chosen and a user-defined intensity threshold was applied to select clusters and avoid their coalescence. For quantification of gephyrin or GABA_AR synaptic clusters, gephyrin or receptor clusters comprising at least 3 pixels and colocalized on at least 1 pixel with VGAT clusters were considered. The number of clusters, the surface area and the integrated fluorescence intensities of clusters were measured. For surface expression analysis, quantification was performed using ImageJ (National Institutes of Health and LOCI, University of Wisconsin). Several dendritic regions of interest were manually chosen and mean average intensity per pixel was measured.