PSD-95 overexpression correlates with increased nNOS expression at synapses. (A) Immunostaining against nNOS (red) and PSD-95 (blue) and their colocalization (white) obtained in the same field of stratum radiatum of a control hippocampal slice culture. Note the existence of PSD-95 puncta devoid of nNOS staining as well as nNOS puncta negative for PSD-95. (B) Same as in A but in a field containing a dendritic segment from a PSD-95–EGFP-transfected neuron (green). Colocalization between nNOS and PSD-95 immunostainings or nNOS and PSD-95–EGFP fluorescence are shown in white. Note the enlargement of the PSD-95 puncta and the presence of large nNOS-positive spots associated with the PSD-95 puncta. (C) Same as in A but in a field containing a dendritic segment from a PDZ₂ mutant PSD-95–EGFP-transfected neuron. Note the reduced size of nNOS puncta associated with PSD-95–EGFP staining. (D) Same as in A but in a field containing a dendritic segment from a cell cotransfected with PSD-95–EGFP and nNOS siRNA. Note again the reduced nNOS staining associated with PSD-95 puncta. (E) Analysis of the size of nNOS puncta measured as the number of stained pixels under the different conditions analyzed. Data are mean ± SEM (error bars) of the analysis of four different slice cultures (75–105 puncta analyzed per condition; *, P < 0.05; Mann-Whitney test). (F) Colocalization between nNOS and PSD-95 immunostaining (or PSD-95–EGFP fluorescence for the PDZ₂ condition) measured in 9–21 different fields (30 × 30 μm) obtained from four different slice cultures per condition (*, P < 0.05; Mann-Whitney test). Data are mean ± SEM (error bars). Bars, 2 μm.