Physical exercise in combination with cognitive training is known to enhance synaptic plasticity, learning, and memory and lower the risk for various complex diseases including Alzheimer’s disease. Here, we show that exposure of adult male mice to an environmental enrichment paradigm leads to enhancement of synaptic plasticity and cognition also in the next generation. We show that this effect is mediated through sperm RNA and especially miRs 212/132. In conclusion, our study reports intergenerational inheritance of an acquired cognitive benefit and points to specific miRs as candidates mechanistically involved in this type of transmission.

In this study, we demonstrate that exposure of adult mice to EE significantly enhances hippocampal LTP and cognitive function in their offspring. We show that this phenotype is due to changes in the RNA composition in the sperm of the corresponding fathers and identify microRNAs (miR) 212/132 as one factor involved in process.

An environmental factor that was shown to lower the risk for various complex diseases, including those affecting the brain, is the combination of physical exercise and cognitive training, also called environmental enrichment (EE). EE is known to enhance synaptic plasticity in rodents and humans and is thus considered a suitable strategy to reduce the risk for dementia and other cognitive diseases (). Importantly, there is evidence that exposure of juvenile mice to EE can enhance hippocampal synaptic plasticity in their offspring (). Whether EE training in adulthood might also affect synaptic function of the next generation has not been tested so far, and the underlying mechanisms of transgenerational transmission are still poorly understood. There is, however, evidence that RNA in gametes could play a role ().

There is emerging evidence that exposure to environmental stimuli can initiate processes that transmit information to the next generation via non-genetic mechanisms (). Such forms of inter- or transgenerational inheritance have been described for aversive stimuli, such as chronic or early life stress that lead to altered response of the hypothalamic-pituitary-adrenal axis, increased anxiety and depressive-like behavior in the following generations (). There is also evidence that exposure of individuals to detrimental environmental stimuli can lead to cellular adaptations that protect the offspring when they are exposed to the same environmental insult (). The idea that environmental factors can affect germ cells and thereby alter biological processes in the offspring is fascinating and may play an important role in the pathogenesis of complex diseases, especially in neuropsychiatric disorders ().

The born mice were subjected to behavioral testing when they were adult. In accordance with our previous observations on LTP enhancement, the mice that originated from oocytes injected with EE sperm RNA showed improved memory function in the FC ( Figures S6 E and S6F, p = 0.04, t test) and water maze paradigms ( Figures S6 G and S6H, platform crossings: p = 0.01, Mann-Whitney test; relative time of platform occupancy: p = 0.04, t test.) when compared to those that originated from oocytes injected with HC sperm RNA. This observation was further corroborated when we analyzed the cognitive score as described above ( Figures 4 E and 4F), suggesting that similar to the intergenerational inheritance of cognitive enhancement mediated by EE, injection of EE sperm RNA into fertilized oocytes also results in a cognitive benefit. In contrast to its effect on LTP, the miR212/132 cluster appeared to have no effect on the behavioral readout. Mice that were born from oocytes injected with EE sperm RNA and co-injected with miR212/132 inhibitors exhibited a similar, albeit insignificant, trend for memory enhancement ( Figures 4 E and S6 F–S6H; percentage time freezing: p = 0.28, Mann-Whitney test; platform crossings: p = 0.19, Mann-Whitney test; relative time of platform occupancy: p = 0.08, t test). These findings suggest that the intergenerational effect of EE on LTP and memory enhancement critically depends on sperm RNA and that the LTP effect is mediated via altered levels of miR212/132. In contrast, miR212/132 levels cannot explain the enhanced memory function indicating that additional mechanisms contribute to the intergenerational enhancement of learning behavior in response to EE. Finally, miR212/132 are not upregulated in the offspring of EE fathers ( Figure S8 ), which suggests that the mechanisms underlying EE-mediated enhanced synaptic plasticity and cognition in the F0 and F1 generation are likely to be different. This interpretation could also explain why the effect of EE in our study is inter- and not transgenerational.

Next, we decided to address the question of whether sperm RNA and in particular miR212/132 would play a role in the memory enhancement seen in the offspring born to EE fathers. As described for the LTP experiments (see Figure 3 ). we isolated RNA from the sperm of EE mice and injected this RNA into fertilized oocytes with scrambled RNA. Oocytes injected with RNA from sperm of HC mice together with scrambled RNA were used as control. Also, here, we included a group where EE sperm RNA was co-injected with miR212/132 inhibitors ( Figure 4 D).

First, we tested mice in the contextual FC. We observed that mice born to EE fathers showed elevated freezing behavior when compared to mice born to HC fathers ( Figures S6 A and S6B, t test, p < 0.05 for HC:HC versus EE:HC group). Similar results were observed in the water maze paradigm for platform crossings and relative time of platform occupancy ( Figures S6 C and S6D, t test p < 0.05, HC:HC versus EE:HC group). We also employed a more stringent approach using a linear-mixed model for statistical analysis taking into account the effect of the different litters, since for the behavioral experiments we employed more than one mouse/litter. In this analysis, we observed a non-significant trend for memory enhancement in the offspring of EE fathers in the FC and water maze paradigms (percentage of time freezing: t = 1.45, df = 10, p = 0.17; platform crossings: t = 1.79, df = 10, p = 0.1; relative time of platform occupancy: t = 1.99, df = 10, p = 0.07). In sum, these data suggest that the intergenerational effect of EE on memory function is subtler compared to the LTP phenotype. This may in part be due to the fact that the analysis of LTP in hippocampal slice preparation allows for a well-defined and specific readout, while the analysis of an animal’s complex behavior as an estimate of memory function, on the other hand, offers a rather limited dynamic range. Rather than relying on the classical readout of the FC and water maze paradigms, we therefore decided to also calculate an integrated cognitive score based on a principal component analysis (PCA). The parameters included in the integrated cognitive score were (1) relative time of platform occupancy in MWM, (2) number of platform crossings in MWM and (3) percentage of time freezing in FC. The first component of the PCA analysis captures the most variance, and hence we took the scores from the PC1 (principal component 1) as a “cognitive score” that would reflect the overall change in cognitive function. This score revealed a significant difference between animals born to HC and EE fathers ( Figures 4 B and 4C linear mixed model t-value = 2.80, df = 10, p = 0.018), confirming that there is indeed intergenerational inheritance of a mild, but significant cognitive advantage after EE exposure in the fathers.

Next, we wondered whether this enhancement of LTP was accompanied by an improvement in cognitive performance. We therefore subjected the offspring of EE fathers and those of HC controls to behavioral testing ( Figure 4 A). We observed no difference in explorative behavior and basal anxiety among groups ( Figure S4 ). Next, mice were subjected to two hippocampus-dependent behavioral tests, namely, the contextual fear-conditioning (FC) and Morris water maze (MWM) paradigms. To avoid ceiling effects on learning, mice were trained using rather mild protocols. Thus, the electric footshock applied during FC was 0.5 mA, an intensity that allows the detection of memory improvement. Of note, there was no difference between the groups in pain sensation as measured by their reaction to the footshock ( Figure S4 E). In the MWM, mice were trained for a maximum of 5 days, a protocol that in our hands results in a moderate memory consolidation of the platform location in wild-type (WT) animals, which normally form robust spatial memory only after 8 training days ( Figure S5 ).

(F) Plot illustrating the magnitude of change in the different groups of each individual parameter of the cognitive score. Significance for the offspring of oocyte injections was calculated using a two-tailed Student’s t test (see Experimental Procedures ).

(D) Oocyte injection scheme. The injections were carried out as described in Figure 2 . The mice born from these injected fertilized oocytes were then tested in behavioral tasks at the age of 3–4 months.

(B) Mice born to EE fathers have a significantly bigger cognitive score. Significance for the F1 generation was calculated using linear mixed models to account for batch and litter effects (see Experimental Procedures ).p < 0.05 (t-value = 2.80, df = 10). HC:HC: n = 29, N = 6; EE:HC: n = 32, N = 7 (n represents number of mice, N represents number in litter).

We observed that the offspring born to oocytes injected with RNA from 10-week EE mice exhibited enhanced LTP, which was reversed to control (HC) levels if miR212/132 inhibitors were co-administered ( Figures 3 C and 3D). These data demonstrate that EE in adult males enhances hippocampal synaptic plasticity in their offspring and that this effect is mediated through sperm RNA causally involving miR212/132.

Since the mothers were never exposed to EE, which might have affected maternal care, the enhanced synaptic plasticity observed in the offspring of fathers that underwent EE training must be associated with changes in the father’s gametes. Previous reports linked sperm RNA to transgenerational inheritance and observed, for example, that individual miRs were altered in sperm of mice that passed an acquired anxiety phenotype to the next generation (). Apart from this, there is additional evidence that manipulating miRs levels in gametes can alter the offspring’s phenotype (). At the same time, miRs are known to play key roles in promoting synaptic plasticity (). We therefore hypothesized that miRs might play a role in the intergenerational transmission of EE-induced LTP enhancement. First, we measured the sperm microRNAome detectable in mice used in our experimental system. Out of 1,886 miRs present in the mouse genome, 219 were identified in sperm ( Figures 2 A and 2B ). We subjected these 219 miRs detected in sperm to a PubMed search according to the following criteria: (1) expression in the brain, (2) having been linked to brain plasticity and memory function, and (3) having a documented role in brain development since this might be a possible route of action by which miRs present in gametes could affect brain function in the offspring ( Figure 2 B). Using these criteria, we identified 6 miRs that showed more than 1 PubMed hit, namely, miR212/132, let-7d, let-7c, let-7b, miR34c, and miR124. MiR212/132 was the top hit according to our search criteria ( Figures 2 B and 2C). These miRs are co-expressed from the same locus, have been shown to affect synaptic function and learning behavior in mice, and play a role in brain development (). Therefore, we assayed the expression of the miR212/132 cluster in mice upon EE training. We found that miR132 and miR212 were upregulated both in sperm and hippocampus of mice that were exposed to EE for 10 weeks ( Figures 3 A and 3B ). Of note, a shorter duration of EE (2 weeks) was not sufficient to induce the upregulation of these miRNAs in sperm ( Figure S2 A), while increased levels were observed in the hippocampus ( Figure S2 B). Furthermore, none of the other miRs we had identified as candidates for the observed intergenerational phenotypes (let-7b, let-7c, let-7d, miR34c, and miR124; see Figure 2 ), nor randomly selected miRs exhibited altered expression after 10 weeks of EE ( Figures S2 C and S2D). These data argue against a general increase of sperm miRs in response to EE and led us to hypothesize that miR212/132 might play a role in the intergenerational transmission of the EE phenotype. To test this possibility, we first injected RNA from sperm of HC or EE mice into fertilized oocytes and examined LTP in the corresponding offspring once they were adult. RNA in both groups was co-injected with scrambled RNA allowing us to include a third group in which we injected into oocytes sperm RNA from EE mice along with miR212/132 inhibitor, which we had previously validated for its inhibitory action ( Figure S3 ).

(C) Oocyte injection scheme. Sperm RNA from a pool of HC or EE mice was isolated and mixed with scrambled negative control (vehicle) or miR212/132 inhibitors and injected into the cytoplasm of fertilized oocytes. Control (“HCi + vehicle”) oocytes were injected with sperm RNA from HC mice + scrambled negative control. “EEi + vehicle” oocytes were injected with sperm RNA from EE mice + scrambled negative control. “EEi + miRNA212/132 inhibitors” oocytes were injected with RNA from EE mice + miR212/132 inhibitors. The mice born from these injected fertilized oocytes were used for LTP measurements at the age of 3–4 months.

miR212/132 Are Increased in the Brain and Sperm of EE Males and They Are Involved in Intergenerational Inheritance of the Enhanced LTP Phenotype

Figure 3 miR212/132 Are Increased in the Brain and Sperm of EE Males and They Are Involved in Intergenerational Inheritance of the Enhanced LTP Phenotype

(C) Graph showing the 6 candidate miRNAs and the corresponding number of hits based on the PubMed search. PubMed IDs for the papers linked to miR212/132 are 22845676, 22246100, 19557767, 23520022, 27392631, 20613834; let-7d are 23425148, 21307844, 20557304, 25799420; let-7c are 25962166, 21676127; let-7-b are 21676127, 27539004; miR34c are 26402112, 21946562; miR124 are 24784359, 22837048.

(B) Venn diagram showing that, out of 1,886 miRs analyzed, 219 were expressed in sperm. This list of miRs was then searched for miRs implicated in brain function and development, synaptic plasticity, and learning and memory (PubMed search criteria “brain + learning + microRNA-X (let-X)”; more than 1 hit). This approach revealed 6 candidate miRs.

First, we wanted to confirm that our EE protocol enhances hippocampal LTP in adult mice. To this end, we subjected mice to 10 weeks of EE training before measuring hippocampal LTP at the Schaffer Collateral CA1 synapse. We observed a highly significant increase in LTP in EE mice compared to home-caged (HC) controls ( Figure 1 A). Next, we wanted to test whether EE training in adult male mice would affect synaptic plasticity in their offspring (F1 generation), which are not subjected to EE. To this end, adult male mice underwent 10 weeks of EE training and were then mated to HC females ( Figure 1 B). We measured hippocampal LTP when the offspring were adult (3 months of age). Notably, offspring of EE fathers had increased LTP compared to those born to fathers that were housed in HC (controls; Figure 1 C). The effect was similar in both male and female offspring ( Figure S1 ). In order to account for possible confounding factors, we performed a linear regression model including the effect of paternal treatment (HC versus EE), sex (male versus female), and paternal treatment X sex interaction. In line with our previous analysis, we observed a highly significant effect of paternal treatment (p < 0.0001). We then tested whether this phenotype is passed on to the grandoffspring of the original EE animals, representing the F2 generation. Here, we did not observe any difference in the LTP between the groups ( Figure 1 D). These data indicate that EE in male mice leads to enhanced hippocampal synaptic plasticity in offspring, but that this effect represents inter- (and not trans-) generational inheritance.

(B) Mating scheme: mice are subjected to EE for 10 weeks and mated, and the offspring are tested 3–4 months after birth. Controls spend the same amount of time in the HC.

Discussion

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Mansuy I.M. Potential of environmental enrichment to prevent transgenerational effects of paternal trauma. Our data demonstrate that EE during adulthood mediates the enhancement of hippocampal LTP in the adult offspring. This is not only the first confirmation, but also an important extension to the original observation by, who showed that EE in juvenile mice (2 weeks of age) enhances LTP in their offspring. Our observation that this phenomenon occurs even when EE is initiated at a time point at which brain development is complete has important implications, suggesting that exercise before conception could provide a brain plasticity benefit to the offspring. Interestingly, Arai et al. observed that EE-mediated intergenerational enhancement of LTP is transmitted through the mothers but not the fathers. This can be explained by the fact that the authors subjected mice to EE before sexual maturity (i.e., at 14 days of age) for 2 weeks, when female gametes are already present. In contrast sperm production begins only at sexual maturity when mice are 6–8 weeks of age. These facts can plausibly explain why 2 weeks of EE in males starting at the age of 14 days failed to elicit intergenerational transmission. Thus, in our study EE was initiated when mice were 10 weeks of age and thus sexually mature. Moreover, we provide evidence that, in addition to enhanced LTP, offspring of EE fathers exhibit a mild but significant cognitive advantage when compared to offspring of HC fathers. When compared to the LTP effect, the memory enhancement was, however, moderate. One explanation could be that the analysis of Schaffer collateral CA1 LTP in hippocampal slice preparation is much more sensitive and thus better suited to detect increased or decreased plasticity compared to behavioral assays that offer a limited dynamic range for the detection of changes. In line with this, calculation of a combined cognitive score on the basis of two hippocampal memory tests using PCA showed that offspring born to EE fathers or from oocytes treated with RNA isolated from sperm of EE mice exhibit a significant cognitive benefit. It also has to be considered that in our study we analyzed memory function in healthy WT mice. It will thus be interesting to see whether EE training in fathers would provide a benefit in synaptic plasticity and memory function to offspring in a situation when brain plasticity is challenged as it is for example the case during aging or in neurodegenerative conditions. In support of this view, recent findings show that EE partially ameliorates the detrimental transgenerational effects observed in offspring born to parents that were exposed to stressful experiences (). Similarly, it will be interesting to see whether increased brain plasticity is observed in offspring when the mother undergoes EE training. In this study, we did not address this issue and rather focused on EE fathers since studying the corresponding gametes for subsequent mechanistic analysis is more suited for an initial study. It is, moreover, important to notice that the male mice used for mating were removed from the cage upon conception and thus never came to contact with the offspring. It is thus highly unlikely that the described phenotypes in offspring are due to differences in maternal care. Further support for this view stems from the observation that mice born from fertilized oocytes that were injected with sperm RNA of EE mice recapitulate the phenotype observed in the offspring of EE fathers. Moreover, offspring born to enriched parents and raised by a HC housed dam also display enhanced memory formation ( Figure S7 ). It will be important for future research to test whether EE training of adult female mice will also transmit a synaptic plasticity and cognitive benefit to offspring and to elucidate the underlying mechanisms. Especially interesting will be to see whether EE induces upregulation of miR212/132 in oocytes and whether this or other processes play a role in the intergenerational transmission of EE-mediated enhanced synaptic plasticity and cognition. Such experiments have to take into account a number of additional issues such as the fact that most protocols to collect oocytes include a superovulation regime that could potentially confound oocyte plasticity such as miR expression.

In conclusion, the idea that EE training in adulthood provides a cognitive benefit not only to the individual undergoing this procedure, but also to its offspring is fascinating. Whether these findings are translatable to humans needs to be determined. Nevertheless, the accumulating evidence that sperm RNA content encodes information about environmentally induced phenotypic traits is an issue that not only needs to be considered in reproductive medicine, but may also offer the chance to discover biomarkers for complex diseases.