the density of GIRK1, GIRK2 and GIRK3 at extrasynaptic sites in dendritic spines of CA1 pyramidal cells in the adult, quantification of immunolabeling was performed from 60 μm coronal slices processed for pre-embedding immunogold in three different layers: the proximal stratum radiatum (defined as the portion in the 100 μm away from the stratum pyramidale), distal stratum radiatum (defined as the portion in the 100 μm away from the border of the stratum lacunosum–moleculare) and stratum lacunosum–moleculare. For each of three animals from different postnatal ages and adult, three samples of tissue were obtained. Randomly selected areas were then photographed from the selected ultrathin sections at a final magnification of 45 000×. Quantification of immunogold labelling was carried out in reference areas totalling approx. 2000 μm2 for each age. Immunoparticles identified in dendritic spines were counted and the surface area of each spine was measured. The data (density of GIRK subunits in spines in each CA1 layer) were expressed as the number of immunoparticles/μm2. We calculated the non-specific labelling density in every reaction in the nuclei of pyramidal cells, a subcellular compartment that should not contain any GIRK. The immunoparticle density over the nuclei was 0.04 ± 0.01 immunoparticles/μm2.
