Reversing Behavioral, Neuroanatomical, and Germline Influences of Intergenerational Stress

Abstract

BACKGROUND:Stressors affect populations exposed to them as well as offspring. Strategies preventing the intergenerational propagation of effects of stress would benefit public health. Olfactory cue-based fear conditioning provides a framework to address this issue. METHODS:We 1) exposed adult male mice to an odor, acetophenone (Ace) or Lyral (parental generation [F0]-Exposed), 2) trained mice to associate these odors with mild foot shocks (F0-Trained), and 3) trained mice to associate these odors with mild foot shocks and then extinguished their fear toward these odors with odor-only presentations (F0-Extinguished). We then examined sensitivity of future generation (F1) offspring to these odors, expression of M71 odorant (Aceresponsive) and MOR23 odorant (Lyral-responsive) receptor-expressing cell populations in F1 offspring, and DNA methylation at genes encoding the Ace-(Olfr151, Olfr160) and Lyral-(Olfr16) responsive receptors in F0 sperm. RESULTS:Extinguishing fear toward Ace or Lyral of F0 male mice (F0-Extinguished) that had been fear conditioned with Ace or Lyral, respectively, results in F1-Extinguished offspring that do not demonstrate behavioral sensitivity to Ace or Lyral, respectively, and do not have enhanced representation for M71 or MOR23 odorant receptors in the olfactory system, as is observed in F1-Trained-Ace or F1-Trained-Lyral cohorts, respectively. The promoters of genes encoding Olfr151 and Olfr160 receptors are less methylated in F0-Trained-Ace sperm compared with F0-Exposed-Ace sperm. The Olfr16 promoter is less methylated in F0-Trained-Lyral sperm compared with F0-Exposed-Lyral sperm, and F0Extinguished-Lyral sperm have methylation levels comparable to F0-Exposed-Lyral sperm. CONCLUSIONS:Our study demonstrates the potential of using extinction-based behavioral strategies to reverse influences of parental stress in offspring and in the parental germline.

Proximate causes and consequences of intergenerational influences of salient sensory experience

“…Training mice to associate a specific odor (eg, Odor A) with a mild foot‐shock results in these mice developing fearful behavior toward Odor A and possessing increased neuroanatomical expression of odorant receptors that detect Odor A . From an intergenerational perspective, we have demonstrated that after mice have been exposed to Odor A + shock conditioning their naïve offspring possess a sensitivity to Odor A and increased neuroanatomical expression of odorant receptors that detect Odor A . Ours are not the only studies to have reported intergenerational imprints of parental olfactory experience.…”

“…F0‐Lyral treatment: Due to the position of MOR23‐GFP glomeruli on the olfactory bulb it is challenging to visualize and quantitate their size and instead we used western blotting to measure the GFP levels as we have performed in our previous publication (Figure S3B). We found a significant increase in GFP expression in the MOE of F1‐trained Lyral F0 sperm RNA animals compared with F1‐exposed Lyral F0 sperm RNA animals ( F = 7.19, P = .021, d R = 1.201) (Figure G,H) (full blots shown in Figure S6).…”

“…In our previous studies, we reported alterations in DNA methylation at odorant receptor loci of the F0 sperm genome. As we noted in those publications, we did not subscribe to the view that these changes in methylation were in any way causal to the effects in offspring but rather discussed these alterations as evidence for the germline registering a salient environmental experience.…”

“…Many cases of such intergenerational influences of sensory environments arise via social transmission through maternal behavior and via the transfer of chemical cues across maternal‐fetal barriers or egg barriers in viviparous or oviparous species, respectively . While there is accumulating evidence that intergenerational influences of sensory environments are observed in offspring of worms, fruit flies and mice that were not conceived at the time of parental exposure to salient sensory stimuli, mechanisms underlying these influences are unknown. To the best of our knowledge, our study is the first to show that RNA found in the male germline of mammals exposed to salient olfactory experience can contribute to impacting olfactory‐related neuroanatomy and function in future generations.…”

“…We measured baseline behavioral sensitivity of adult mice derived from RNA‐injected zygotes to odors using an odor potentiated startle (OPS) behavioral assay that we and others have used previously and that measures acoustic startle response to a noise burst. Animals were habituated to the startle chambers for 5‐10 minutes on three separate days.…”