Transgenerational inheritance of behavioral and metabolic effects of paternal exposure to traumatic stress in early postnatal life: evidence in the 4th generation

Abstract

In the past decades, evidence supporting the transmission of acquired traits across generations has reshaped the field of genetics and the understanding of disease susceptibility. In humans, pioneer studies showed that exposure to famine, endocrine disruptors or trauma can affect descendants, and has led to a paradigm shift in thinking about heredity. Studies in humans have however been limited by the low number of successive generations, the different conditions that can be examined, and the lack of mechanistic insight they can provide. Animal models have been instrumental to circumvent these limitations and allowed studies on the mechanisms of inheritance of environmentally induced traits across generations in controlled and reproducible settings. However, most models available today are only intergenerational and do not demonstrate transmission beyond the direct offspring of exposed individuals. Here, we report transgenerational transmission of behavioral and metabolic phenotypes up to the 4th generation in a mouse model of paternal postnatal trauma (MSUS). Based on large animal numbers (up to 124 per group) from several independent breedings conducted 10 years apart by different experimenters, we show that depressive-like behaviors are transmitted to the offspring until the third generation, and risk-taking and glucose dysregulation until the fourth generation via males. The symptoms are consistent and reproducible, and persist with similar severity across generations. These results provide strong evidence that adverse conditions in early postnatal life can have transgenerational effects, and highlight the validity of MSUS as a solid model of transgenerational epigenetic inheritance.

Keywords: 3rd and 4th generation; behavior; early-life trauma; epigenetic inheritance; mouse model; transgenerational.

Figure 1:

MSUS paradigm. MSUS consists of (A) separating mouse pups (F1) from their mother (F0, naïve primiparous control females mated with naïve males) daily for 3 hours per day at an unpredictable time during the 12 hours active cycle, starting 1 day after birth (postnatal day 1, PND1) until PND14. (B) During separation, dams are exposed to an additional unpredictable stressor by being subjected to either, a forced swim in 18°C water for 5 minutes or a 20-minute physical restraint in a tube, anytime (unpredictably) during the 3 hours. From PND15, mice are left undisturbed with their mother until PND21 (no further MSUS), are then weaned at PND21 and raised normally until adulthood (C). Control litters are raised normally (left). Males used to generate the pups are removed from the breeding cage shortly after mating thus, fathers never encounter their offspring and do not contribute to their rearing. When adult (3–8 months of age), F1 males are paired with naïve primiparous control females to sire the F2 generation, then F2 and F3 males are bred with naïve primiparous control females to generate an F3 and F4 offspring, respectively. Males from each generation are tested on the elevated plus maze, forced swim test, weight measurements and glucose response after physical restraint. MSUS is applied only to F1 mice, mice from F2, F3 and F4 generations are not exposed to any manipulation. Phenotypes transmitted from father to offspring are intergenerational, phenotypes that persist from father to offspring then grand-offspring or great grand-offspring are transgenerational

Figure 2:

persistent behavioral effects of MSUS across 3 generations on the elevated plus maze. MSUS treatment (A) increases the amount of time spent on the open arms of an elevated plus maze in F1 and F2 mice but not F3 mice (F1 control n = 124, MSUS n = 118, t₂₄₀ = 2.26 P = 0.025; F2 control n = 49, MSUS n = 45, t₉₂ = 2.096 P = 0.039; F3 control n = 38, MSUS n = 57, t₉₃ = 1.244 P = 0.217) and (B) decreases the latency to first enter an open arm in F1, F2 and F3 mice (F1 control n = 111, MSUS n = 101, t₂₁₀ = 5.298 P < 0.0001; F2 control n = 41, MSUS n = 39, t₇₈ = 4.353 P < 0.0001; F3 control n = 40, MSUS n = 59, t₉₇ = 3.51 P = 0.0007). Data represent median ± whiskers. Reported n represents data after outlier removal using the ROUT test at Q = 5%. *P < 0.05, ***P < 0.001, ****P < 0.0001

Figure 3:

reproducible behavioral alterations by MSUS in F3 and F4 generations. Depressive-like symptoms shown by increased time spent floating on a forced swim test in MSUS males from (A) F3 generation but not from (C) F4 generation (F3: control n = 20, MSUS n = 19 t₃₇ = 3.37 P = 0.0018; F4: Batch 2 control n = 24, MSUS n = 26 t₄₈ = 0.424 P = 0.6732). In (B), separate batches of F4 males (Batches 1 and 2) were tested on the elevated plus maze. Time spent on the open arms and latency to first enter an open arm are similarly altered in both batches. (Batch 1 for time spent on open arms: control n = 22, MSUS n = 19, t₅₂ = 2.49 P = 0.0161; Batch 1 for latency to enter an open arm: control n = 22, MSUS n = 19, t₅₁ = 2.432 P = 0.019; Batch 2 for spent time on open arms: control n = 27, MSUS n = 27, t₃₉ = 2.159 P = 0.037; Batch 2 latency to first enter an open arm: control n = 28, MSUS n = 25, t₃₉ = 2.209 P = 0.033). Data represent median ± whiskers. Reported n represents data after outlier removal using the ROUT test at Q = 5%. *P < 0.05, **P < 0.01

Figure 4:

behavioral phenotypes in F4 female progeny. (A) F4 MSUS females do not significantly differ from control females in time spent floating during the forced swim test (control n = 20, MSUS n = 14, t₃₂ = 0.918 P = 0.366). (B) Time spent in the open arms of the elevated plus maze was increased (control n = 24, MSUS n = 20, t₄₂ = 2.09 P = 0.043), while latency to first enter an open arm was decreased in F4 MSUS females (control n = 20, MSUS n = 16, t₃₄ = 3.01 P = 0.005). Data represent median ± whiskers. Reported n represents data after outlier removal using the ROUT test at Q = 5%. *P < 0.05, **P < 0.01

Figure 5:

transgenerational effects of MSUS treatment on glucose level. (A) Baseline glucose was measured in whole blood following tail prick in F3 (left) and F4 males (right) (F3 control n = 13, MSUS n = 18, t₂₉ = 1.891 P = 0.069; F4 control n = 8, MSUS n = 8, t₁₄ = 1.84 P = 0.087). Continuing from (A), glucose concentrations in F4 blood (B) was measured at 15-minute intervals during a 30-minute physical restraint challenge, and 60 minutes after release from the restraint tube (control n = 8, MSUS n = 8, for interaction F_{3, 42} = 2.99 P = 0.042). (C) Body weight of F4 males was measured at PND1, PND21 and in adulthood (PND1: control n = 48, MSUS n = 52, t₉₉ = 1.29 P = 0.199; PND21: control n = 46, MSUS n = 50, t₉₄ = 2.27 P = 0.025; adult: control n = 46, MSUS n = 51, t₉₅ = 1.86 P = 0.065). (D) Food intake (control n = 11, MSUS n = 12, t₂₁ = 2.185 P = 0.04; n represents number of cages) and (E) tail length were measured in adult mice (control n = 46, MSUS n = 51, t₉₂ = 1.38 P = 0.172). Data represent median ± whiskers, except for (B) which represent mean ± s.e.m. Reported n represents data after outlier removal. #P < 0.1, *P < 0.05