Alex fluor 555

E16.5 inner ears or eyes were fixed for 1 hour in PFA 4%, equilibrated in sucrose 20% overnight and embedded in a 1:1 mixture of sucrose 20%: OCT (Tissue-Tek). Cryosections (12μm) were stored at −80C, thawed, and processed for citrate antigen retrieval (boiled five times for 1 minute in 10mM citrate with cooling intervals). The sections were then permeabilized and blocked in 0.5% Triton X-100 and 1% BSA, and incubated with the primary antibodies overnight (NR2F1: R&D Systems PPH813299, mouse monoclonal clone H8132 (1:100); SOX2: Santa Cruz Biotechnology sc-17320, goat polyclonal (1:100)). Secondary antibodies were donkey anti-mouse conjugated to Alex Fluor 555 and donkey anti-goat conjugated to Alex Fluor 647 (Thermofisher). Nuclei were stained with Hoechst 33342 (Thermofisher). For the quantifications in Fig. 3D, a 25×25 μm region of interest was defined to match the organ of Corti, and signal intensity was measured separately for the SOX2 and NR2F1 channels using ImageJ (IntDens). For whole-mounts at birth (Fig. 5), inner ears were fixed 1 hour in 4% PFA, the sensory epithelia were exposed, permeabilized in 0.5% Triton X-100, stained with phalloidin conjugated to Alexa Fluor 488 (Thermofisher), and mounted flat. Images were acquired with a Zeiss LSM800 confocal microscope.

To examine the neural activity of BLA-projecting vHPC neurons during Exp OF, we bilaterally injected Cholrea Toxin Subunit B (CTB) conjugated to AlexFluor-555 (200 nL per side, 100 nL / min, ThermoFisher Scientific) in BLA aimed at the following coordinates relative to bregma: AP: −1.40 mm; ML: ±3.40 mm; DV: −5.00 mm. Mice were allowed to recover for 3 days before returning to group housing with cagemates. On Day 1, observers were subjected to CFC. On Day 2, we then tested observers for Exp OF with a familiar demonstrator (W/+/+) or with stranger demonstrator (W/+/−). 1 hour after behavioral testing, mice were perfused and immunostained for Arc and NeuN. We collected 2–4 vHPC coronal sections (AP; −3.20 mm) per mouse. In Figure 7B, we examined a total of 12,872 (W/+/−: 5,943, W/+/+: 6,929) NeuN⁺ cells. The percentage of total Arc⁺CTB⁺ neurons was calculated out of total CTB⁺ cells ( $\frac{\mathit{\text{Total Arc}}+\mathit{\text{CTB}}+\mathit{\text{cells}}}{\mathit{\text{Total CTB}}+\mathit{\text{cells}}}*100\text{\%}$ ). Fold change analysis (normalized by the average of the control W/+/− group) was compared between groups. The percentage of total Arc⁺CTB⁻ neurons was calculated out of total CTB⁻ cells ( $\frac{\mathit{\text{Total Arc}}+\mathit{\text{CTB}}-\mathit{\text{cells}}}{\mathit{\text{Total CTB}}-\mathit{\text{cells }}}*100\text{\%}$ ). Fold change analysis (normalized by the average of the control W/+/− group) was compared between groups. Quantification of NeuN, CTB, and Arc positive cells in vHPC was conducted using the Cell Counting plugin in ImageJ.