Parental olfactory experience influences behavior and neural structure in subsequent generations

Journal name:
Nature Neuroscience
Volume:
17,
Pages:
89–96
Year published:
DOI:
doi:10.1038/nn.3594
Received
Accepted
Published online
Corrected online

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.

At a glance

Figures

  1. Behavioral sensitivity to odor is specific to the paternally conditioned odor.
    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, F2,30 = 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, F2,26 = 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.

  2. Sensitivity of F1 males toward F0-conditioned odor.
    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).

  3. Neuroanatomical characteristics of the olfactory system in F1 males after paternal F0 olfactory fear conditioning.
    Figure 3: Neuroanatomical characteristics of the olfactory system in F1 males after paternal F0 olfactory fear conditioning.

    (af) 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, F2,91 = 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, F2,84 = 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, F2,13 = 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.

  4. Behavioral sensitivity and neuroanatomical changes are inherited in F2 and IVF-derived generations.
    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). (cf). 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.

  5. Behavioral sensitivity and neuroanatomical changes persist after cross-fostering.
    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, F3,18 = 6.874, planned post hoc comparisons). (cf) 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, F3,14 = 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, F3,15 = 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.

  6. Methylation of odorant receptor genes in sperm DNA from conditioned F0 and odor naive F1 males.
    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.

  7. Experimental design to investigate the effect of cue-specific trauma to an F0 generation on subsequently conceived generations.
    Supplementary Fig. 1: Experimental design to investigate the effect of cue-specific trauma to an F0 generation on subsequently conceived generations.

    Novel experimental paradigm that uses olfactory fear conditioning to examine the structural and functional changes in the nervous systems of adult mice (F1 and F2) conceived after the F0 generation was trained to associate specific odorant presentations with mild foot-shocks. Briefly, F0 adult male mice were trained to associate Acetophenone or Propanol presentation with mild-footshocks (5 odor-shock pairings/session, 3 sessions, 1 session/day). Ten days after this conditioning, these F0 males were mated with naïve females. Ten days after the mating was setup, the F0 males were separated from the females. F1 offspring born were tested at 2-months of age. For studies of the F2 generation, F1 males that had no previous exposure to either Acetophenone or Propanol were mated with naïve females for 10 days, and resulting F2 offspring were used for analyses. Our experimental design minimized the possibility of a “social transmission” mode of information transfer. Specifically, the F0 male has absolutely no contact with the F1 offspring, is placed with the female 10 days after the last conditioning day, should not have any trace of the conditioned odor on his skin or hair to transfer to the mother, and is separated from the female after a 10 day period to minimize any in utero exposure of the pups to the conditioned male.

  8. No differences found in anxiety measures in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol.
    Supplementary Fig. 2: No differences found in anxiety measures in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol.

    C57Bl/6J (a) and M71-LacZ (b) adult male offspring (F1-Home, F1-Ace, F1-Prop) spend the same amount of time in the closed and open arms of an elevated plus maze, and make the same number of entries into the open arms. (C57Bl/6J: F1-Home-C57 n = 9 vs F1-Ace-C57 n = 8) (F1-Home-M71 n =11 vs F1-Ace-M71 n =12 vs F1-Prop-M71 n =11) (Two-way ANOVA: p > 0.05 in both experiments).

  9. No differences in auditory fear conditioning in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol.
    Supplementary Fig. 3: No differences in auditory fear conditioning in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol.

    No significant differences were found between F1-Home-C57, and F1-Ace-C57 in the acquisition (a), consolidation (b), and extinction retention (c) of the memory of an aversive auditory cue after they were trained to associate 6kHz tone presentations with mild-footshocks. (C57Bl/6J: F1-Home-C57 n = 9 vs F1-Ace-C57 n = 9) p > 0.05 in all experiments.

  10. Cross-fostering study to determine transmission versus inheritance of observed effects.
    Supplementary Fig. 4: Cross-fostering study to determine transmission versus inheritance of observed effects.

    Sexually naïve C57Bl/6J female mice were conditioned with Acetophenone or left in their Home Cage; they were then mated with C57Bl/6J males for 10 days. Offspring were then divided into the following groups: Offspring of Home Cage Mothers (F1-Home-C57), Offspring of Acetophenone Conditioned Mothers (F1-Ace-C57), O-MHC cross fostered starting at P1 by Mothers Conditioned to Acetophenone (F1-Home-C57fostered), O-MCA cross fostered by Home Cage Mothers (F1-Ace-C57fostered).

  11. Bisulfite sequencing around the Olfr151 and Olfr6 genes was conducted to query the methylation status of CpG di-nucleotides.
    Supplementary Fig. 5: Bisulfite sequencing around the Olfr151 and Olfr6 genes was conducted to query the methylation status of CpG di-nucleotides.

    Coding sequences (red text) of Olfr6 and Olfr151 in reverse complement with primers used to generate amplicons highlighted in blue. CpG di-nucleotides shown in Fig. 6 are numbered. The CpG di-nucleotides not numbered in Olfr151 could not be queried due to technical issues.

  12. Methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE DNA of the odor naive F1 and F2 generations.
    Supplementary Fig. 6: Methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE DNA of the odor naive F1 and F2 generations.

    (a) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE of the F1 generation male reveals no differences in methylation between groups (p > 0.05) (n = 4/group). (b) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE of the F1 generation reveals no differences in methylation at individual CpG sites between groups. (c) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 gene in MOE of the F2 generation reveals no differences in methylation status between F2-Ace and F2-Prop (n = 4/group) across all CpG di-nucleotides queried (p > 0.05). (d) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 gene in MOE of the F2 generation reveals no differences in methylation status between F2-Ace and F2-Prop across specific CpG di-nucleotides queried (Bonferroni corrected for multiple comparisons). All graphs represent Mean±SEM.

  13. Validation of Sperm N-ChIP protocol, and histone modifications around the M71 locus in the sperm of F0 males (fathers) that had been subjected to olfactory fear conditioning.
    Supplementary Fig. 7: Validation of Sperm N-ChIP protocol, and histone modifications around the M71 locus in the sperm of F0 males (fathers) that had been subjected to olfactory fear conditioning.

    (a) Sperm ChIP was validated by performing qPCR for Pold3 and Bmp4. As has been shown previously, Pold3 is associated with more of the “activating” mark (Acetyl H3), and less of the “repressive” mark (H3 trimethyl-K27), while BMP4 is associated with more of the repressive than the activating mark. N-ChIP on sperm of F0 males (fathers) conditioned either to Acetophenone (F0-Ace-C57) or Propanol (F0-Prop-C57) reveals no significant differences in the activating (Acetyl H3) (b) or repressive (H3 trimethyl-K27) (c) histone modifications immunoprecipitated in our experiment. (n = 5 epidydymis per sample, n = 3 samples/group) (p > 0.05 for both marks). All graphs represent Mean±SEM.

Change history

Corrected online 09 December 2013
In the version of this article initially published online, the base grant to the Yerkes National Primate Research Center was omitted from the Acknowledgments. The error has been corrected for the print, PDF and HTML versions of this article.

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Affiliations

  1. Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia, USA.

    • Brian G Dias &
    • Kerry J Ressler
  2. Yerkes National Primate Research Center, Atlanta, Georgia, USA.

    • Brian G Dias &
    • Kerry J Ressler
  3. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • Kerry J Ressler

Contributions

B.G.D. conceived of the project, designed and performed experiments, analyzed the data, and wrote the paper. K.J.R. obtained funds, designed experiments, analyzed the data, wrote the paper and supervised the project.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Experimental design to investigate the effect of cue-specific trauma to an F0 generation on subsequently conceived generations. (64 KB)

    Novel experimental paradigm that uses olfactory fear conditioning to examine the structural and functional changes in the nervous systems of adult mice (F1 and F2) conceived after the F0 generation was trained to associate specific odorant presentations with mild foot-shocks. Briefly, F0 adult male mice were trained to associate Acetophenone or Propanol presentation with mild-footshocks (5 odor-shock pairings/session, 3 sessions, 1 session/day). Ten days after this conditioning, these F0 males were mated with naïve females. Ten days after the mating was setup, the F0 males were separated from the females. F1 offspring born were tested at 2-months of age. For studies of the F2 generation, F1 males that had no previous exposure to either Acetophenone or Propanol were mated with naïve females for 10 days, and resulting F2 offspring were used for analyses. Our experimental design minimized the possibility of a “social transmission” mode of information transfer. Specifically, the F0 male has absolutely no contact with the F1 offspring, is placed with the female 10 days after the last conditioning day, should not have any trace of the conditioned odor on his skin or hair to transfer to the mother, and is separated from the female after a 10 day period to minimize any in utero exposure of the pups to the conditioned male.

  2. Supplementary Figure 2: No differences found in anxiety measures in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol. (53 KB)

    C57Bl/6J (a) and M71-LacZ (b) adult male offspring (F1-Home, F1-Ace, F1-Prop) spend the same amount of time in the closed and open arms of an elevated plus maze, and make the same number of entries into the open arms. (C57Bl/6J: F1-Home-C57 n = 9 vs F1-Ace-C57 n = 8) (F1-Home-M71 n =11 vs F1-Ace-M71 n =12 vs F1-Prop-M71 n =11) (Two-way ANOVA: p > 0.05 in both experiments).

  3. Supplementary Figure 3: No differences in auditory fear conditioning in adult male offspring that had been conceived after the F0 generation males had been subjected to olfactory fear conditioning with acetophenone or propanol. (74 KB)

    No significant differences were found between F1-Home-C57, and F1-Ace-C57 in the acquisition (a), consolidation (b), and extinction retention (c) of the memory of an aversive auditory cue after they were trained to associate 6kHz tone presentations with mild-footshocks. (C57Bl/6J: F1-Home-C57 n = 9 vs F1-Ace-C57 n = 9) p > 0.05 in all experiments.

  4. Supplementary Figure 4: Cross-fostering study to determine transmission versus inheritance of observed effects. (45 KB)

    Sexually naïve C57Bl/6J female mice were conditioned with Acetophenone or left in their Home Cage; they were then mated with C57Bl/6J males for 10 days. Offspring were then divided into the following groups: Offspring of Home Cage Mothers (F1-Home-C57), Offspring of Acetophenone Conditioned Mothers (F1-Ace-C57), O-MHC cross fostered starting at P1 by Mothers Conditioned to Acetophenone (F1-Home-C57fostered), O-MCA cross fostered by Home Cage Mothers (F1-Ace-C57fostered).

  5. Supplementary Figure 5: Bisulfite sequencing around the Olfr151 and Olfr6 genes was conducted to query the methylation status of CpG di-nucleotides. (376 KB)

    Coding sequences (red text) of Olfr6 and Olfr151 in reverse complement with primers used to generate amplicons highlighted in blue. CpG di-nucleotides shown in Fig. 6 are numbered. The CpG di-nucleotides not numbered in Olfr151 could not be queried due to technical issues.

  6. Supplementary Figure 6: Methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE DNA of the odor naive F1 and F2 generations. (78 KB)

    (a) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE of the F1 generation male reveals no differences in methylation between groups (p > 0.05) (n = 4/group). (b) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 (M71) gene in MOE of the F1 generation reveals no differences in methylation at individual CpG sites between groups. (c) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 gene in MOE of the F2 generation reveals no differences in methylation status between F2-Ace and F2-Prop (n = 4/group) across all CpG di-nucleotides queried (p > 0.05). (d) Bisulfite sequencing data to query the methylation status of CpG di-nucleotides in the Olfr151 gene in MOE of the F2 generation reveals no differences in methylation status between F2-Ace and F2-Prop across specific CpG di-nucleotides queried (Bonferroni corrected for multiple comparisons). All graphs represent Mean±SEM.

  7. Supplementary Figure 7: Validation of Sperm N-ChIP protocol, and histone modifications around the M71 locus in the sperm of F0 males (fathers) that had been subjected to olfactory fear conditioning. (93 KB)

    (a) Sperm ChIP was validated by performing qPCR for Pold3 and Bmp4. As has been shown previously, Pold3 is associated with more of the “activating” mark (Acetyl H3), and less of the “repressive” mark (H3 trimethyl-K27), while BMP4 is associated with more of the repressive than the activating mark. N-ChIP on sperm of F0 males (fathers) conditioned either to Acetophenone (F0-Ace-C57) or Propanol (F0-Prop-C57) reveals no significant differences in the activating (Acetyl H3) (b) or repressive (H3 trimethyl-K27) (c) histone modifications immunoprecipitated in our experiment. (n = 5 epidydymis per sample, n = 3 samples/group) (p > 0.05 for both marks). All graphs represent Mean±SEM.

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  1. Supplementary Text and Figures (31,422 KB)

    Supplementary Figures 1–7

Additional data