The olfactory critical period is determined by activity-dependent Sema7A/PlxnC1 signaling within glomeruli

Six-week-old mice used in this assay were unilaterally naris-occluded at P0 and the occluded naris was reopened at P6 or P10. Mice without occlusion (–) were analyzed as controls. Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Next, a filter paper spotted with the 1 st odor was presented three times (detection trials 4–6). Then, a filter paper spotted with the 2nd odor was presented three times (detection trials 7–9). Investigation times (s) observed during each presentation were measured. Odorant pairs examined were as follows: top, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and bottom, 62 mM (+)-CAR and 64 mM EUG. CAR, carvone; EUG, eugenol. The average values of investigation times are shown.

Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Then, a filter paper spotted with 0.5 μl of 20 mM VNL or 6.4 M eugenol (EUG) was presented three times (detection trials 4–6). Investigation times (s) observed during each presentation were measured. Mice were conditioned to VNL at P2~4 or P9~11, and were analyzed as adults at 6 weeks (6w). Mice without VNL conditioning (–) were analyzed as controls. Odorants with 10⁻³ dilution were also analyzed. The average values of investigation times are shown.

Individual glomeruli possess unique but different levels of Sema7A expression determined by intrinsic activity of ORs, forming the glomerular rank of Sema7A expression. OB sections were immunostained with anti-Sema7A antibodies. Intensities of Sema7A signals were determined for each glomerulus and plotted in order. Glomerular rank of fluorescent signals is shown for 437 different glomeruli in the OB at P8. Expression levels of Sema7A are indicated for the rI7, MOR29A, VNL-stimulated (P5~7) MOR29A, and CNG^- rI7 glomeruli.

M/T cells at P5 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 M/T cells with one primary dendrite (mature) and those with multiple branched dendrites (immature) are compared in the rI7 glomeruli: CNG⁺, 14/17 (82.3 %); CNG^-, 3/14 (21.4 %) in (A), Tg-Sema7A, CNG⁺, 15/18 (83.3 %); Tg-Sema7A, CNG^-, 13/16 (81.3 %) in (B), and Tg-Sema7A (Y213S), CNG⁺, 3/13 (23.0 %); Tg-Sema7A (Y213S), CNG^-, 2/9 (22.2 %) in (C).

Mice were habituated to the cage and then a filter paper spotted with 0.5 μl of distilled water was presented for 3 min. This was repeated three times with 1 min intervals (control trials 1–3). Next, a filter paper spotted with the 1st odor was presented three times (detection trials 4–6). Then, a filter paper spotted with the 2nd odor was presented three times (detection trials 7–9). Investigation times for odors were measured in the PlxnC1 cKO and WT male mice at 6w. Odorant pairs examined are as follows: left, 6.2 M (+)-CAR and 6.4 M EUG; middle, 6.2 M (+)-CAR and 6.3 M (–)-CAR; and right, 62 mM (+)-CAR and 64 mM EUG. CAR, carvone; EUG, eugenol. The average values of investigation times are shown.

Pups of the WT and PlxnC1 cKO were exposed to VNL at P2~4 or P9~11 and analyzed at 6 w. Immediately after the transfer to a new cage, a filter paper spotted with VNL was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without VNL exposure (–) were analyzed as negative controls. PPA was used as an attractive-odor control (gray). Temperature differences before (T) and after (Tx) the transfer are compared. Error bars are SD (n = 3, 4, 4, 3, 3 animals). *p<0.05; ***p<0.005 (Student’s t-test). VNL, vanillin; PPA, propionic acid.

Sniffing times (s) were measured in the habituation/dishabituation test. The WT and Oxt KO were conditioned to 4MT at P2~4. Mice without conditioning (–) were analyzed as negative controls (n = 4 animals). Odorants used were as follows: 100 mM and 10 μM for 4MT (top); 6.4 M and 6.4 mM for EUG (bottom). The average values of total sniffing times are shown.

Pups of the WT and Oxt KO were exposed to 4MT at P2~4 and analyzed at 6w. Immediately after the transfer to a new cage, a filter paper spotted with 4MT was presented to the mice. The rectal temperature was measured every 20 min in each mouse during the test. Unconditioned mice without 4MT exposure (–) were analyzed as negative controls. Temperature differences before (T) and after (Tx) the transfer are compared. 4MT, 4-methyl-thiazole; EUG, eugenol.

The KO mice were administrated with oxytocin (Oxt) or saline (SAL) by intraperitoneal injection at P0~6 (A) or P8~14 (B). Time duration of sniffing are shown after the presentations of the same unfamiliar female mouse (trials 1–6). Data also show mean ± standard error.