Knockdown and pharmacological blockade of nNOS prevents MIS formation. (A) Contour representation (top) and 3D reconstruction (bottom) of a dendritic segment obtained from a cell cotransfected with PSD-95 and an siRNA against nNOS (siNOS). Note the small number of MISs (top; numbers refer to presynaptic terminals [*] making synaptic contacts on the spine) and the persistence of larger spines (top and bottom). (B) Same as in A but for a PSD-95–transfected cell treated for 2 d with 200 μM of the NOS inhibitor L-NAME. L-NAME prevented MIS formation (top) but not the PSD-95–induced spine and PSD enlargement (bottom). (C) Quantitative analysis illustrating the proportion of MISs detected under control conditions (ctrl; n = 8 cells; 145 spines) and in cells transfected with EGFP (n = 4 cells; 164 spines), PSD-95 (n = 7 cells; 234 spines), PSD-95 and siNOS (n = 5 cells; 127 spines), and PSD-95 treated with L-NAME (n = 4 cells; 128 spines). Both siNOS transfection and L-NAME treatment significantly prevented MIS formation (*, P < 0.05 vs. PSD-95). (D) Same as in C but for the spine volume. siNOS transfection and L-NAME treatment did not prevent PSD-95–induced spine enlargement (*, P < 0.05 vs. control). (E) Same as in C but for the total PSD area. siNOS transfection and L-NAME treatment only partially reduced the PSD enlargement associated with PSD-95 overexpression (*, P < 0.05 vs. control). Data are mean ± SEM (error bars). Bars: (A and B, top) 5 μm; (A and B, bottom) 1 μm.