Figure 2. Long-term asymmetric expression of N-cadherin induces elimination of glutamatergic synapses.

(A, B) Immunocytochemical stainings for VAMP2 and fluorescence images of coexpressed PSD-95-EGFP in N-cadherin knockout neurons (Ncad−/−) and in N-cadherin knockout neurons expressing N-cadherin (Ncad−/− + N-cadherin) 2 days (A) and 8 days (B) after transfection at 14 DIV. Segments of proximal dendrites are shown. Scale bars: 2,5 µm. Upper panels are fluorescence images and lower panels are thresholded (black or white) images used to quantify puncta. Dendrites were identified using the PSD-95-EGFP images. Merge: Overlay of thresholded VAMP2 (red) and PSD-95-EGFP (green) puncta; >80% of PSD-95 puncta colocalized with VAMP2. (C, E, F) Quantification of dendritic density of VAMP2 puncta (including few autaptic puncta, C), VAMP2 puncta area (E), and VAMP2 puncta average intensity (F). −/−: N-cadherin knockout neurons; Ncad: N-cadherin knockout neurons expressing N-cadherin. (G) Quantification of dendritic density of PSD-95-EGFP puncta. (H) Relative changes of dendritic density of glutamatergic synapses (colocalized VAMP2 and PSD-95-EGFP puncta, excluding autaptic puncta) in N-cadherin knockout neurons expressing N-cadherin. Normalized to values of N-cadherin knockout neurons (control) at matched times in culture. Note that selectively at 8 days after transfection (8d, 22 DIV), but not at 2 days after transfection (2d, 16 DIV), the dendritic density of synapses is reduced. (D) Quantification of dendritic density of VAMP2 puncta 2 days (2d) and 8 days (8d) after transfection at an earlier stage in culture (9–11 DIV). GFP: control N-cadherin knockout neurons. Ncad: N-cadherin knockout neurons expressing N-cadherin. Note the reduced increase in synapse density in N-cadherin knockout neurons expressing N-cadherin at 8 days after transfection. Means ± SEM. n (cells) is indicated on bars. *, P<0.05; **, P<0.01 and P<0.001 (in C, G) Students t-test.