(A) Sema7A expression stimulated by VNL exposure. The ECFP-tagged MOR29A glomeruli were analyzed by immunohistochemistry after the exposure to VNL at P2~4. OB sections were immunostained at P4 using antibodies against Sema7A. Relative fluorescent signals within the MOR29A glomeruli were analyzed in the mice with (+) or without (–) VNL exposure. **p<0.01 (Student’s t-test). Error bars are standard deviation, SD (n = 6 animals for each condition). See also Figure 3—figure supplement 1A. (B) MOR29A glomeruli in the VNL-exposed mice. Left: Changes in the levels of synapse markers. Pups were exposed to VNL at different time periods, and the OB sections at P11 were immunostained for pre- and post-synapse markers, vGlut2 and GluR1, respectively. Signal intensities within the glomerular layer (GL) were normalized by those detected in the olfactory nerve layer (ONL) or external plexiform layer (EPL), and compared with the VNL-unexposed controls (–). **p<0.01 (Student’s t-test). n.s., not significant. Error bars indicate SD (n = 6, 3, 3, 5, 5 glomeruli for each condition). See also Figure 3—figure supplement 1B. Right: Dendrite selection within the MOR29A glomeruli. The mice conditioned to VNL (P2~4) and unconditioned (–) were analyzed. M/T cells at P4 were visualized by Lucifer yellow (LY) injection into the glomeruli (Figure 3—figure supplement 2). Intracellular LY injection was performed as previously described (Inoue et al., 2018). The numbers of M/T cells with one dendrite (mature) and those with multiple dendrites (immature) were counted in the MOR29A glomeruli. The ratios (%) of mature (dark blue) and immature (cyan) M/T cells are shown: VNL-cond., 12/17 (70.6 %); VNL-uncond., 4/16 (25.0 %). n = 6, 5 glomeruli. See also Figure 3—figure supplement 2. (C) VNL-responding cells connecting to the MOR29A glomeruli. Left: Detection of tufted and periglomerular cells activated by VNL. The MOR29A glomeruli were identified by ECFP signals. The sections were then counter-stained with DAPI. Levels of an immediate-early gene product EGR1 (red) surrounding the MOR29A glomeruli were analyzed at 3w by immunostaining of OB sections (25 μm-thick). The mice were conditioned to VNL at P2~4 or P9~11. Middle: Quantification of EGR1 signals. EGR1 signals in the glomerular layer (GL) and external plexiform layer (EPL) were counted as activated periglomerular cells and tufted cells, respectively. Mice without VNL exposure in neonates were analyzed as controls (–). Error bars are SD (n = 4, 4, 5 animals). The one-way ANOVA was applied on values. ***p<0.005 (Student’s t-test). n.s., not significant. Scale bar is 30 μm. Right: MOR29A-positive OSNs. ECFP-tagged MOR29A OSNs in the OE were counted at P21 after the exposure to VNL at P2~4 or P9~11. To identify the MOR29A-positive OSNs, OE sections were immunostained with antibodies against GFP. Relative numbers of MOR29A+ OSNs are compared for the mice with (P2~4, 9~11) or without (–) VNL exposure. n.s., not significant. Error bars are standard deviation, SD (n = 6, 5, 4 animals). See also Figure 3—figure supplement 1C. (D) Glomerular sizes with or without VNL conditioning. Relative sizes (ratios of diametral areas) of the MOR29A glomeruli were measured at 3w after the VNL exposure at different time periods in neonates. Glomeruli without VNL exposure (–) were analyzed as negative controls. Error bars are SD (n = 6, 3, 3, 5, 5 animals). VNL, vanillin; GL, glomerular layer; ONL, olfactory nerve layer; EPL, external plexiform layer.