Olfactory fear conditioning leads to an increase in conditioned-odor-responsive cells in parents (F0) that is heritable (F1).

(A) Schematic representation of the mouse main olfactory epithelium and olfactory bulb. MOE: main olfactory epithelium. OB: olfactory bulb. (B) Timeline of olfactory fear conditioning and MOE collection. (C) Experimental paradigms for olfactory fear conditioning groups. Mice in the paired condition received a foot shock that co-terminated with odor presentation, while mice in the unpaired condition received a foot shock 60 seconds after odor presentation. (D) Schematic demonstrating the process by which cells of interest in the MOE were quantified. Epithelia from both M71-IRES-tauGFP+/+ and MOR23-IRES-tauGFP+/+ adult mice were cleared using the iDISCO+ tissue-clearing protocol. Samples were imaged on a light sheet microscope and analyzed using Imaris spot detection software. (E) Images of the MOE before (left) and after (right) optical tissue clearing. (F) The average number of M71 olfactory sensory neurons in a 3503 μm3 cube of the epithelium in the propanol unpaired (light blue) and propanol paired (dark blue) conditions (Student’s unpaired t-test. Unpaired vs. paired P=0.3009. n=6,7.). (G) Example images of M71 OSNs in zone 1 of cleared MOE from both the unpaired (left) and paired (middle) conditions. Example image of an MOE with the counted cells represented by colored dots (right). Each set of colors represents a distinct counting cube. Scale bar: 200μm. (H) Graph showing the differences between the average number of M71 OSNs in a 3503 μm3 cube of epithelium of naive (gray), acetophenone unpaired (lighter green), and acetophenone paired (darker green) conditions in F0 and F1 (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 paired P<0.0001. F0 unpaired vs. paired P<0.0001. Naïve vs. F1 paired P<0.0001. F1 unpaired vs. paired P<0.0001. n=12,11,12,12,14.). (I) Example images of MOR23 OSNs in zone 1 of cleared MOE from both the unpaired (left) and paired (middle) conditions. Example image of an MOE with the counted cells represented by colored dots (right). Scale bar: 200μm. (J) Graph showing the differences between the average number of MOR23 OSNs in a 3503 μm3 cube of epithelium in naive (gray), lyral unpaired (lighter purple), and lyral paired (darker purple) conditions in F0 and F1 (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 paired P=<0.0001. F0 unpaired vs. paired P<0.0001. F1 unpaired vs. paired P=0.0368. n=7,9,9,6,6.).

Conditioned-odor-responsive cell increase is sustained through at least 9 weeks of cell turnover.

(A) Timeline of olfactory fear conditioning and extended MOE collection time points. (B) The average number of M71 OSNs in a 3503 μm3 cube of epithelium of unpaired (light green) and paired (dark green) mice, 42-or 63-days post-conditioning (One-way ANOVA. P=0.0033. Tukey’s multiple comparisons. 42d unpaired vs. paired P=0.0476. 63d unpaired vs. paired P=0.0011. n=8,8,4,6.).

Olfactory fear conditioning biases olfactory receptor choice toward conditioned-odor-responsive cell-specific identities.

(A) Timeline of olfactory fear conditioning, EdU injections, and MOE collection. (B) Schematic representation of the distinct layers of the MOE, showing the stem cell, immature OSN, and mature OSN populations (left). Representative image of the MOE from an MOR23GFP+/+ mouse showing EdU-positive cells (red) and a newborn (EdU+) MOR23 OSN (green). Scale bar: 20 μm. (C) Representative images showing staining of EdU (red, first column), endogenous GFP (green, second column), DAPI (blue, third column), and the merged channels (fourth column) in both M71GFP+/+ and MOR23GFP+/+ MOE. Scale bar: 40μm. (D) Percentage of EdU-positive M71 OSNs in naïve, unpaired, and paired groups (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. paired P<0.0001. Unpaired vs. paired P<0.0001. n=6,6,6.). (E) Percentage of EdU-positive MOR23 OSNs in naïve, unpaired, and paired groups (One-way ANOVA. P=0.0120. Tukey’s multiple comparisons. Naïve vs. paired P=0.0154. Unpaired vs. paired P=0.0653. n=4,6,8.).