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, breeding for the F1 generation, 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) 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. (G) The average number of M71 OSNs in a 3503 µm3 cube of epithelium of naïve (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.). Squares indicate males, triangles indicate females. (H) 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. (I) The average number of MOR23 OSNs in a 3503 µm3 cube of epithelium in naïve (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.).

Increased OSN abundance is specific to OSN subtypes responsive to the conditioned odor.

(A) Timeline of olfactory fear conditioning and MOE collection for both experiments. (B) M71-IRES-tauRFP2 targeted mutation. (C) Representative images showing DAPI (blue, top left), endogenous RFP in M71 OSNs (red, top right), endogenous GFP in MOR23 OSNs (green, bottom left), and the merged channels (bottom right) in an M71-RFP+/+;MOR23GFP+/+ animal. Scale bar: 50µm. (D) 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.). Squares indicate males, triangles indicate females. (E) The ratio of M71 OSNs to 100 MOR23 OSNs in M71-RFP+/+;MOR23GFP+/+ mice following no conditioning (naïve, gray), unpaired (light green), or paired (dark green) olfactory fear conditioning with acetophenone (One-way ANOVA. P=0.0062. Tukey’s multiple comparisons. Naïve vs. paired P=0.0097. Unpaired vs. paired P=0.0163. n=2,4,4.).

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 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 (magenta) and a newborn (EdU+) MOR23 OSN (green and magenta). Scale bar: 20 µm. (C) Representative images showing MOE 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+/+ mice. Scale bar: 40µm. (D) Percentage of EdU-positive M71 OSNs in naïve, unpaired, and paired groups (Kruskal-Wallis test. P<0.0001. Dunn’s multiple comparisons. Naïve vs. paired P=0.0009. Unpaired vs. paired P=0.0799. n=6,6,6.). Squares indicate males, triangles indicate females. (E) Percentage of EdU-positive MOR23 OSNs in naïve, unpaired, and paired groups (Kruskal-Wallis test. P=0.0003. Dunn’s multiple comparisons. Naïve vs. paired P=0.0040. Unpaired vs. paired P=0.0725. n=4,6,8.).

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) Example image of M71 OSNs in zone 1 of cleared MOE collected 42 days post-conditioning. Scale bar: 200µm. (C) 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=0.0203. n=8,8,4,6.). Squares indicate males, triangles indicate females. (D) Schematic of the trichamber behavioral approach-avoidance assay and equation for the approach-avoid index. Positive values indicate approach to the conditioned odor (acetophenone), while negative values indicate avoidance. (E) The approach-avoid indices of unpaired and paired F0 mice at day 42 (Student’s unpaired t-test. 42d unpaired vs. paired P=0.2248. n=10,11.). Squares indicate males, triangles indicate females. (F) The approach-avoid indices of unpaired and paired F0 mice at day 63 (Student’s unpaired t-test. 63d unpaired vs paired P=0.8987. n=6,8.).

Olfactory fear conditioning leads to nuanced behavioral differences in F1 offspring.

(A) Schematic of the trichamber assay showing the three conditioning odors and control odors. Propanol was the control odor for acetophenone and lyral, and acetophenone was the control odor for propanol. (B) Timeline of olfactory fear conditioning, behavior testing in F0, F0 breeding for the F1 generation, and F1 behavior testing. Conditioned F0 males used to breed for F1s did not undergo behavioral testing to prevent any extinction effects. (C) The approach-avoid indices of acetophenone-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 Paired P<0.0001. F0 Unpaired vs. F0 Paired P<0.0001. F0 Paired vs. F1 Unpaired P<0.0001. F0 Paired vs. F1 Paired P<0.0001. n=25,10,15,22,18.). Squares indicate males, triangles indicate females. (D) Group-averaged heat maps for acetophenone-conditioned F0 mice and F1 offspring, with the acetophenone chamber on the left and the control (propanol) chamber on the right. (E) Distance traveled in the trichamber assay for acetophenone-conditioned F0 mice and F1 offspring (One-way ANOVA. P=0.0041. Tukey’s multiple comparisons. Naïve vs. F0 Paired P=0.0032. F0 Paired vs. F1 Unpaired P=0.0141. F0 Paired vs. F1 Paired P=0.0103. n=25,10,15,22,18). (F) Time freezing for acetophenone-conditioned F0 mice and F1 offspring (Kruskal-Wallis test. P=0.0022. Dunn’s multiple comparisons. F0 Unpaired vs. F1 Unpaired P=0.0406. F0 Paired vs. F1 Unpaired P=0.0262. n=25,10,15,22,18.). (G) The approach-avoid indices of lyral-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 Paired P=0.0004. F0 Unpaired vs. F0 Paired P<0.0001. F0 Paired vs. F1 Unpaired P=0.0013. F0 Paired vs. F1 Paired P<0.0001. n=10,17,20,6,13.). (H) Group-averaged heat maps for lyral-conditioned F0 mice and F1 offspring, with the lyral chamber on the left and the control (propanol) chamber on the right. (I) Distance traveled in the trichamber assay for lyral-conditioned F0 mice and F1 offspring (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F1 Paired P<0.0001. F0 Unpaired vs. F1 Unpaired P=0.037. F0 Unpaired vs. F1 Paired P<0.0001. F0 Paired vs. F1 Unpaired P=0.0121. F0 Paired vs. F1 Paired P<0.0001. F1 Unpaired vs. F1 Paired P=0.0325. n=10,17,20,6,13.). (J) Time freezing for lyral-conditioned F0 mice and F1 offspring (Kruskal-Wallis test. P<0.0001. Dunn’s multiple comparisons. Naïve vs. F0 Unpaired P=0.0001. Naïve vs. F0 Paired P=0.0005. F0 Unpaired vs. F1 Paired P=0.0012. F0 Paired vs. F1 Paired 0.0041. n=10,17,20,6,13.). (K) The approach-avoid indices of propanol-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 Paired P<0.0001. F0 Unpaired vs. F0 Paired P<0.0001. F0 Paired vs. F1 Unpaired P<0.0001. F0 Paired vs. F1 Paired P<0.0001. n=25,13,18,13,13.). (L) Group-averaged heat maps for propanol-conditioned F0 mice and F1 offspring, with the propanol chamber on the left and the control (acetophenone) chamber on the right. (M) Distance traveled in the trichamber assay for propanol-conditioned F0 mice and F1 offspring (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 Unpaired P=0.0304. Naïve vs. F0 Paired P<0.0001. F0 Unpaired vs. F1 Unpaired P=0.0014. F0 Paired vs. F1 Unpaired P<0.0001. F1 Unpaired vs. F1 Paired P=0.0045. n=25,13,18,13,13.). (N) Time freezing for propanol-conditioned F0 mice and F1 offspring (Kruskal-Wallis test. P<0.0001. Dunn’s multiple comparisons. Naïve vs. F0 Unpaired P=0.0074. Naïve vs. F0 Paired P<0.0001. F0 Paired vs. F1 Unpaired P=0.0011. F0 Paired vs. F1 Paired P=0.028. n=25,13,18,13,13.).

Unsupervised machine learning analysis identifies behavioral differences in F1.

(A) Schematic of the trichamber assay showing the three conditioning odors and control odors. Propanol was the control odor for acetophenone and lyral, and acetophenone was the control odor for propanol. (B) Analysis pipeline for Keypoint-MoSeq. Eight keypoints were tracked across the entire 10-minute trichamber assay for 99 videos, and the tracking data was used to train a Keypoint-MoSeq model. (C) Trajectory plots of the 21 most frequently used syllables across the dataset. (D) The relative usage frequencies of syllables in the whole trichamber arena in the F1 offspring of unpaired (light pink) and paired (dark pink) fathers. Syllables with significantly different usage between groups are underlined in the x-axis and denoted with asterisks above the data points (Kruskal-Wallis test with Dunn’s multiple comparisons. Syllable 3 F1 unpaired vs. F1 paired P=0.00696. Syllable 5 P=0.0054. Syllable 11 P=0.02505. Syllable 22 P=0.0184. n=35,34.). (E) Histogram of the frequency (number of observations divided by the bin width) of each animal’s center point along the x-axis (x distance from the conditioned odor port). The two vertical bars indicate the divisions between the three chambers (conditioned odor chamber left, control odor chamber right). (F) The relative usage frequencies of syllables in the conditioned odor chamber in F1 unpaired (light pink) and F1 paired (dark pink) (Kruskal-Wallis test with Dunn’s multiple comparisons. Syllable 5 F1 unpaired vs. F1 paired P=0.0406. Syllable 17 P=0.046695. n=35,34.). (G) The relative usage frequencies of syllables in the whole arena in the F1 offspring of acetophenone-unpaired (light green) and acetophenone-paired (dark green) fathers (n=18.13.). (H) The relative usage frequencies of syllables in the whole arena in the F1 offspring of lyral-unpaired (light purple) and lyral-paired (dark purple) fathers (Kruskal-Wallis test with Dunn’s multiple comparisons. Syllable 2 lyral F1 unpaired vs. F1 paired P=0.0016. Syllable 3 P=0.02724. Syllable 5 P=0.003. Syllable 7 P=0.00252. Syllable 15 P=0.0208. Syllable 20 P=0.00015. n=6,13.). (I) The relative usage frequencies of syllables in the whole arena in the F1 offspring of propanol-unpaired (light blue) and propanol-paired (dark blue) fathers (Kruskal-Wallis test with Dunn’s multiple comparisons. Syllable 6 propanol F1 unpaired vs. F1 paired P=0.0206. Syllable 12 P=0.04068. Syllable 14 P=0.0072. Syllable 15 P=0.03204. Syllable 20 P=0.0148. n=11,8.).

Conditioned odor-responsive OSN counts by sex.

(A) The average number of M71 OSNs in a 3503 µm3 cube of epithelium of naïve (gray), acetophenone unpaired (lighter green), and acetophenone paired (darker green) conditions in F0 and F1, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.0425. n=5,7,9,2,12,0,5,7,9,5.). (B) The average number of MOR23 OSNs in a 3503 µm3 cube of epithelium of naïve (gray), lyral unpaired (lighter purple), and lyral paired (darker purple) conditions in F0 and F1, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.7206. n=6,1,7,2,7,2,2,4,1,5.).

F1 conditioned odor-responsive OSN counts by litter.

(A) The average number of M71 OSNs in a 3503 µm3 cube of epithelium of the F1 offspring of acetophenone-unpaired (lighter green), and acetophenone-paired (darker green) fathers, separated by litter (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. UP_A vs. P_B P=0.0329. UP_A vs. P_C P=0.0041. UP_A vs. P_E P=0.0001. UP_B vs. P_B P=0.038. UP_B vs. P_C P=0.0043. UP_B vs. P_E P=0.0002. UP_C vs. P_A P=0.046. UP_C vs. P_B P=0.006. UP_C vs. P_C P=0.0015. UP_C vs. P_D P=0.0241. UP_C vs. P_E P<0.0001. P_A vs. P_E P=0.0257. n=4,3,5,4,4,1,1,4.). (B) The average number of MOR23 OSNs in a 3503 µm3 cube of epithelium of the F1 offspring of lyral-unpaired (lighter green), and lyral-paired (darker green) fathers, separated by litter (One-way ANOVA. P=0.0004. UP_A vs. P_A P=0.0008. UP_A vs. P_B P=0.0133. UP_A vs. P_C P=0.002. n=6,3,1,2.).

Extended timepoint OSN counts by sex.

(A) 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, separated by sex (Two-way ANOVA. Treatment factor P=0.0123. Sex factor P=0.9889. n=5,3,6,2,2,2,3,3.).

Extended timepoint trichamber assay by sex.

(A) The approach-avoid indices of female unpaired and paired F0 mice at day 42 (Student’s unpaired t-test. P=0.0055. n=4,4.). (B) The approach-avoid indices of male unpaired and paired F0 mice at day 42 (Student’s unpaired t- test. P=0.6534. n=6,7.). (C) The approach-avoid indices of female unpaired and paired F0 mice at day 63 (Student’s unpaired t-test. P=0.8762. n=3,4.). (D) The approach-avoid indices of male unpaired and paired F0 mice at day 63 (Student’s unpaired t-test. P=0.9859. n=3,4.).

Trichamber assay metrics by sex.

(A) The approach-avoid indices of acetophenone-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.8137. n=25,0,10,0,15,0,10,12,15,3.). (B) Distance traveled in the trichamber assay for acetophenone-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P=0.0049. Sex factor P=0.0261. n=25,0,10,0,15,0,10,12,15,3.). (C) Time freezing for acetophenone-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P=0.0008. Sex factor P=0.5149. n=25,0,10,0,15,0,10,12,15,3.). (D) The approach-avoid indices of lyral-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.9573. n=5,5,9,8,7,13,4,2,6,7.). (E) Distance traveled in the trichamber assay for lyral-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.016. n=5,5,9,8,7,13,4,2,6,7.). (F) Time freezing for lyral-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P=0.0013. Sex factor P=0.0938. n=5,5,9,8,7,13,4,2,6,7.). (G) The approach-avoid indices of propanol-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.4375. n=25,0,12,1,17,1,8,8,16,4.). (H) Distance traveled in the trichamber assay for propanol-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.0005. Tukey’s multiple comparisons. F0 Unpaired male vs. female P= 0.0165. F0 Paired male vs. female P=0.0165. F1 Unpaired male vs. female P=0.0165. F1 Paired male vs. female P=0.0165. n=25,0,12,1,17,1,8,8,16,4.). (I) Time freezing for propanol-conditioned F0 mice and F1 offspring, separated by sex (Two-way ANOVA. Treatment factor P<0.0001. Sex factor P=0.3945. n=25,0,12,1,17,1,8,8,16,4.).

Trichamber assay avoidance index by litter.

(A) The approach-avoid indices of acetophenone-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by litter (One-way ANOVA. P=0.487. n=4,4,9,5,3,2,7,6.). (B) The approach-avoid indices of lyral-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by litter (One-way ANOVA. P=0.9939. n=1,5,9,4.). (C) The approach-avoid indices of propanol-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers, separated by litter (One-way ANOVA. P=0.0199. Tukey’s multiple comparisons. UP_A vs. UP_C P=0.0306. UP_A vs. P_B P=0.0088. UP_A vs. P_C P=0.041. n=2,7,4,1,4,8.).

Additional trichamber assay metrics.

(A) The mean speed of acetophenone-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P=0.003. Tukey’s multiple comparisons. Naïve vs. F0 Paired P=0.0024. F0 Paired vs. F1 Unpaired P=0.0121. F0 Paired vs. F1 Paired P=0.0081. n=25,10,15,22,18.). (B) The number of entries into the propanol (control) and acetophenone (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P=0.0174. Odor factor P=0.1339. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=25,10,15,22,18.). (C) Time spent in the propanol (control) and acetophenone (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P=0.0022. Odor factor P=0.0053. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=25,10,15,22,18.). (D) The mean speed of lyral-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F1 Paired P<0.0001. F0 Unpaired vs. F1 Unpaired P=0.0438. F0 Unpaired vs. F1 Paired P<0.0001. F0 Paired vs. F1 Unpaired P=0.0128. F0 Paired vs. F1 Paired P<0.0001. F1 Unpaired vs. F1 Paired P=0.0315. n=10,17,20,6,13.). (E) The number of entries into the propanol (control) and lyral (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P<0.0001. Odor factor P=0.6465. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=10,17,20,6,13.). (F) Time spent in the propanol (control) and lyral (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P=0.0007. Odor factor P=0.0914. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=10,17,20,6,13.). (G) The mean speed of propanol-conditioned naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (One-way ANOVA. P<0.0001. Tukey’s multiple comparisons. Naïve vs. F0 Unpaired P=0.0295. Naïve vs. F0 Paired P<0.0001. F0 Unpaired vs F1 Unpaired P=0.0017. F0 Paired vs. F1 Unpaired P<0.0001. F1 Unpaired vs. F1 Paired P=0.0051. n=25,13,18,13,13.). (H) The number of entries into the acetophenone (control) and propanol (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P<0.0001. Odor factor P=0.0435. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=25,13,18,13,13.). (I) Time spent in the acetophenone (control) and propanol (conditioned odor) chambers in naïve, unpaired, and paired F0 mice, and F1 mice bred from unpaired and paired F0 fathers (Two-way ANOVA. Treatment factor P=0.4521. Odor factor P=0.0016. P-values from Tukey’s multiple comparisons tests in Table S2.7. n=25,13,18,13,13.).

Density of syllable usage across space in F1 offspring.

(A) Kernel density estimate (KDE) plots of syllable usage as a function of the animal’s center point along the x-axis (x distance from the conditioned odor port). The two vertical bars indicate the divisions between the three chambers (conditioned odor chamber left, control odor chamber right). n=35 (F1 unpaired), 34 (F1 paired).