Parental olfactory experience influences behavior and neural structure in subsequent generations

Abstract

Using olfactory molecular specificity, we examined the inheritance of parental traumatic exposure, a phenomenon that has been frequently observed, but not understood. We subjected F0 mice to odor fear conditioning before conception and found that subsequently conceived F1 and F2 generations had an increased behavioral sensitivity to the F0-conditioned odor, but not to other odors. When an odor (acetophenone) that activates a known odorant receptor (Olfr151) was used to condition F0 mice, the behavioral sensitivity of the F1 and F2 generations to acetophenone was complemented by an enhanced neuroanatomical representation of the Olfr151 pathway. Bisulfite sequencing of sperm DNA from conditioned F0 males and F1 naive offspring revealed CpG hypomethylation in the Olfr151 gene. In addition, in vitro fertilization, F2 inheritance and cross-fostering revealed that these transgenerational effects are inherited via parental gametes. Our findings provide a framework for addressing how environmental information may be inherited transgenerationally at behavioral, neuroanatomical and epigenetic levels.

Figure 1

Behavioral sensitivity to odor is specific to the paternally conditioned odor. (a,b) Responses of individual C57Bl/6J F1 male offspring conceived after the F0 male was fear conditioned with acetophenone. F1-Ace-C57 mice had an enhanced sensitivity to acetophenone (a), but not to propanol (control odor, b) compared with F1-Home-C57 mice (F1-Ace-C57, n = 16; F1-Home-C57, n = 13; t test, P = 0.043, t₂₇ = 2.123). (c,d) Responses of M71-LacZ F1 male offspring conceived after the F0 male was fear conditioned with acetophenone or propanol. F1-Ace-M71 mice had an enhanced sensitivity to acetophenone (c), but not to propanol (d), compared with F1-Home-M71, and F1-Prop-M71 mice. In contrast, F1-Prop-M71 mice had an enhanced sensitivity to propanol (d), but not acetophenone (c) (F1-Home-M71, n = 11; F1-Ace-M71, n = 13; F1-Prop-M71, n = 9; OPS to acetophenone: ANOVA, P = 0.003, F_2,30 = 6.874; F1-Home-M71 versus F1-Ace-M71, P < 0.05; F1-Ace-M71 versus F1-Prop-M71, P < 0.01; OPS to propanol: ANOVA, P = 0.020, F_2,26 = 4.541; F1-Ace-M71 versus F1-Prop-M71, P < 0.05). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01.

Figure 2

Sensitivity of F1 males toward F0-conditioned odor. Association time with either the concentration of odor on the x axis or an empty chamber was recorded. An aversion index was computed by subtracting the amount of time spent in the open chamber from the time spent in the odor chamber. (a) When tested with acetophenone, F1-Ace mice detected acetophenone at a lower concentration (0.03%) than F1-Prop mice, with both groups eventually showing equal aversion at the 0.06% concentration (P = 0.005 with Bonferroni correction for multiple comparisons). (b) When tested with propanol, F1-Prop mice detected propanol at a lower concentration (0.003%) than F1-Ace mice, with both groups eventually showing equal aversion at the 0.006% concentration (P = 0.0005 with Bonferroni correction for multiple comparisons) (F1-Ace-C57, n = 16; F1-Prop-C57, n = 16). Data are presented as mean ± s.e.m. (**P < 0.01).

Figure 3

Neuroanatomical characteristics of the olfactory system in F1 males after paternal F0 olfactory fear conditioning. (a–f) β-galactosidase staining revealed that offspring of F0 males trained to acetophenone (F1-Ace-M71) had larger dorsal and medial acetophenone-responding glomeruli (M71 glomeruli) in the olfactory bulb compared with F1-Prop-M71 and F1-Home-M71 mice. Scale bar represents 1 mm. (g) Dorsal M71 glomerular area in F1 generation (M71-LacZ: F1-Home, n = 38; F1-Ace, n = 38; F1-Prop, n = 18; ANOVA, P < 0.0001, F_2,91 = 15.53; F1-Home-M71 versus F1-Ace-M71, P < 0.0001; F1-Ace-M71 versus F1-Prop-M71, P < 0.05). (h) Medial M71 glomerular area in F1 generation (M71-LacZ: F1-Home, n = 31; F1-Ace, n = 40; F1-Prop, n = 16; ANOVA, P < 0.0001, F_2,84 = 31.68; F1-Home-M71 versus F1-Ace-M71, P < 0.0001; F1-Ace-M71 versus F1-Prop-M71, P < 0.0001). (i) F1-Ace-M71 mice had a larger number of M71 OSNs in the MOE than F1-Prop-M71 and F1-Home-M71 mice (M71-LacZ: F1-Home, n = 6; F1-Ace, n = 6; F1-Prop, n = 4; ANOVA, P = 0.0001, F_2,13 = 18.80; F1-Home-M71 versus F1-Ace-M71, P < 0.001; F1-Ace-M71 versus F1-Prop-M71, P < 0.01). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Behavioral sensitivity and neuroanatomical changes are inherited in F2 and IVF-derived generations. (a,b) Responses of F2-C57Bl/6J males revealed that F2-Ace-C57 mice had an enhanced sensitivity to acetophenone compared with F2-Prop-C57 mice (a). In contrast, F2-Prop-C57 mice had an enhanced sensitivity to propanol compared with F2-Ace-C57 mice (b; F2-Prop-C57, n = 8; F2-Ace-C57, n = 12; OPS to acetophenone: t test, P = 0.0158, t₁₈ = 2.664; OPS to propanol: t test, P = 0.0343, t₁₇ = 2.302). (c–f). F2-Ace-M71 mice whose F0 generation male had been conditioned to acetophenone had larger dorsal and medial M71 glomeruli in the olfactory bulb than F2-Prop-M71 mice whose F0 generation had been conditioned to propanol. Scale bar represents 200 μm. (g) Dorsal M71 glomerular area in F2 generation (M71-LacZ: F2-Prop, n = 7; F2-Ace, n = 8; t test, P < 0.0001, t₁₃ = 5.926). (h) Medial M71 glomerular area in F2 generation (M71-LacZ: F2-Prop, n = 6; F2-Ace, n = 10; t test, P = 0.0006, t₁₄ = 4.44). (i) Dorsal M71 glomerular area in IVF offspring (F1-Prop-IVF, n = 23; F1-Ace-IVF, n = 16; t test, P < 0.001, t₃₇ = 4.083). (j) Medial M71 glomerular area in IVF offspring (F1-Prop-IVF, n = 16; F1-Ace-IVF, n = 19; t test, P < 0.001, t₃₃ = 5.880). Data are presented as mean ± s.e.m.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Behavioral sensitivity and neuroanatomical changes persist after cross-fostering. (a) F1 offspring of mothers that had been fear conditioned with acetophenone (F1-Ace-C57) showed enhanced sensitivity to acetophenone compared with F1-Home-C57 controls (F1-Home-C57, n = 13; F1-Ace-C57, n = 16; t test, P = 0.0256, t₂₇ = 2.362). (b) Cross-fostering behavior. F1-Ace-C57 males had higher OPS to acetophenone than F1-Home-C57 males (P < 0.01). F1-Ace- C57(fostered) males still had higher OPS to acetophenone than F1-Home-C57(fostered) males (P < 0.05) (ANOVA, P = 0.0011, F_3,18 = 6.874, planned post hoc comparisons). (c–f) Cross-fostering neuroanatomy. F1-Ace- M71 males cross-fostered by mothers conditioned to propanol (F1-Ace-M71(fostered)) continued to have larger M71 glomeruli than F1-Prop-M71 males cross-fostered by mothers conditioned to acetophenone (F1-Prop-M71(fostered)). Scale bar represents 100 μm. (g) Dorsal M71 glomerular area in F1 cross-fostered generation (M71-LacZ: F1-Prop, n = 6; F1-Ace, n = 4; F1- Prop(fostered), n = 5; F1-Ace(fostered), n = 3; ANOVA, P < 0.0001, F_3,14 = 17.52; F1-Prop versus F1-Ace, P < 0.001; F1-Prop(fostered) versus F1-Ace(fostered), P < 0.01). (h) Medial M71 glomerular area in F1 cross-fostered generation (M71-LacZ: F1-Prop, n = 4; F1-Ace, n = 3; F1-Prop(fostered), n = 8; F1-Ace(fostered), n = 4; ANOVA, P < 0.01, F_3,15 = 5.933; F1-Prop (fostered) versus F1-Ace(fostered), P < 0.01). Data are presented as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Methylation of odorant receptor genes in sperm DNA from conditioned F0 and odor naive F1 males. (a) Bisulfite sequencing of CpG di-nucleotides in the Olfr151 (M71) gene in F0 sperm revealed that F0-Ace mouse DNA (n = 12) was hypomethylated compared with that of F0-Prop mice (n = 10) (t test, P = 0.0323, t₁₆ = 2.344). (b) A particular CpG di-nucleotide in the Olfr151 (M71) gene in F0 sperm was hypomethylated in F0-Ace mice (n = 12) compared with F0-Prop mice (n = 10) (P = 0.003, Bonferroni corrected). (c) We found no differences in methylation between F0-Ace (n = 12) and F0-Prop (n = 10) mice across all of the CpG di-nucleotides queried in the Olfr6 gene in F0 sperm (P > 0.05). (d) Across specific CpG di-nucleotides in the Olfr6 gene, we found no differences in methylation between F0-Ace (n = 12) and F0-Prop (n = 10) mice (Bonferroni corrected). (e) Bisulfite sequencing of the Olfr151 (M71) gene in F1 sperm revealed that F1-Ace mouse DNA (n = 4) was hypomethylated compared with that of F1-Prop mice (n = 4) (t test, P = 0.0153, t₁₄ = 2.763). (f) Bisulfite sequencing of CpG di-nucleotides in the Olfr151 (M71) gene in F1 sperm revealed that two particular CpG di-nucleotides in the Olfr151 (M71) gene were hypomethylated in F1-Ace mice (n = 4) compared with F1-Prop mice (n = 4) (P = 0.002, Bonferroni corrected). Data are presented as mean ± s.e.m. *P < 0.05 after correction.