PSVue detects PS exposed at spines in the adult OB and DG. (A) Experimental scheme (top) and representative images (bottom) of the GCL of the OB in adult WT mice injected with PSVue480 (green), stained for PSVue550 (red) with or without zinc solution. PSVue480⁺ dots (green arrows) are stained with PSVue550⁺ dots (pink arrows) in a zinc-dependent manner (92.1 ± 1.8% [n = 3 mice, pooled from three independent experiments] of PSVue480⁺ dots were colocalized with PSVue550). (B) Experimental scheme (top) and representative images (bottom) of the GCL of the OB in adult WT mice injected with AnnexinV-FITC (green), stained for PSVue550 (white) and PSD95 (red). Arrows indicate AnnexinV-FITC⁺PSVue550⁺PSD95⁺ spines in the GCL (73.4 ± 1.1% [n = 3 mice, pooled from two independent experiments] of AnnexinV-FITC⁺PSD95⁺ dots were colocalized with PS). (C and D) Representative images of the EPL of the OB (C) or ML of the hippocampus (D) in adult WT mice stained for PSVue480 (PS, white) and Synaptophysin-1 (red in C, top) or PSD95 (red in C, bottom, and D). Arrows indicate double-positive signals (C, 37.9 ± 2.2% and 36.8 ± 1.4% [n = 3 mice, pooled from two independent experiments] of PS⁺ dots is colocalized with Synaptophysin-1 and PSD95, respectively; D, 33.6 ± 0.9% [n = 3 mice, pooled from two independent experiments] of PS⁺ dots is colocalized with PSD95). (E and F) Active apoptosis of tdTomato⁺ granule cells in the adult OB. Representative images of the GCL of the OB in Dcx-CreER^T2; R26-tdTomato mice at 14, 28, and 56 dpi (n = 3 mice) stained for activated caspase-3 (green) are shown. tdTomato⁺ signals (red) are directly observed without staining. Nuclei are stained by Hoechst 33342 (blue). Arrows and arrowheads indicate tdTomato⁺ activated caspase-3⁺ and tdTomato^- activated caspase-3⁺ pyknotic cells, respectively. Proportion of activated caspase-3⁺ cells in tdTomato⁺ cells in the GCL of the adult OB is shown (F; 14 dpi, 4/222 cells from three mice; 28 dpi, 0/546 cells from three mice; 56 dpi, 0/767 cells from three mice; pooled from five independent experiments). (G) PS exposure at spines of nonapoptotic tdTomato⁺ granule cells in the adult OB. Representative images of the GCL of the OB in adult Dcx-CreER^T2; R26-tdTomato mice at 28 dpi (n = 3 mice) stained for PS (white) and activated caspase-3 (green). tdTomato⁺ signals (red) are directly observed without staining. Boxed areas are enlarged in G¹–G³. Arrowheads and arrows indicate activated caspase-3⁺ PS⁺ apoptotic cells and activated caspase-3^- PS⁺ spines, respectively. None of the tdTomato⁺ neurons with exposed PS at their spines expresses activated caspase-3 or PS in their soma at 28 and 56 dpi (n = 27 cells from three mice at 28 dpi; n = 14 cells from three mice at 56 dpi; pooled from four independent experiments). GCL, granule cell layer; EPL, external plexiform layer; ML, molecular layer. Scale bars: A–D, 2 µm; E, 10 µm; G, 5 µm. Data shown are mean ± SEM.