The rostral DG of P32 mice was subjected to serial block-face scanning electron microscopy (sEM). (A, D and G) Coronal sections show part of the granule cell layer (GCL) and the entire width of the ML of the hippocampus of WT (A), Plxna2−/− (D) and Sema5A−/− mice (G). The area boxed in red is shown at higher magnification in panels B and C for WT mice, panels E and F for Plxna2−/− mice, and panels H and I for Sema5A−/− mice. (B, C, E, F, H and I) Examples of higher magnification images of dentate ML, c = capillary. Examples of spine synapses composed of a dendritic spine (s) with a large head, a post-synaptic density (PSD, arrowhead) and a presynaptic bouton (b) with neurotransmitter vesicles. For 3D reconstruction, dendritic segments (each 20–22 µm in length) and their spines were traced in individual sEM images. (J) The number of dendritic protrusions that do not show a PSD is very similar among the three genotypes: WT (0.17 ± 0.03 spines/µm), Plxna2−/− (0.19 ± 0.09 spines/µm) and Sema5A−/− (0.16 ± 0.03 spines/µm) mice. (K) Quantification of spine head volume of WT (0.08 ± 0.01 µm3), Plxna2−/− (0.09 ± 0.01 µm3), and Sema5A−/− (0.07 ± 0.01 µm3) GCs did not reveal a significant difference. (L) Quantification of the surface area of the PSD revealed no significant differences between WT (0.06 ± 0.009 µm2), Plxna2−/− (0.06 ± 0.003 µm2), and Sema5A−/− (0.05 ± 0.003 µm2) mice. (M) The number of presynaptic boutons that contact a dendritic spine (i.e., synapses formed per spine) is not significantly different between WT, Plxna2−/− and Sema5A−/− GCs. (N and O) Two-dimensional EM images of P30 littermate Plxna2+/+ (WT, n = 2) and Plxna2−/− (n = 2) mice (arrow points to PSD). (P) Quantification of synapse density in 2D EM images. Scale bars in A, D, G = 20 µm; in B, E, H = 2 µm, in C, F, I = 0.2 µm, in N, O = 0.8 µm. For quantification of 3D EM images, n = 110, 208, and 165 spines were analyzed for WT (n = 1), Plxna2−/− (n = 1) and Sema5A−/− (n = 1) mice, respectively. Values are represented as mean ± SEM from three to four dendritic segments for each genotype. ***p < 0.001, N.S. = not significant (p > 0.05) two-tailed unpaired Student's t test.