Perinatal environmental enrichment affects murine neonates’ brain structure before their active engagement with environment

Reviewer #1 (Public review):

Kaller et al. (2025) explore the impact of environmental enrichment (EE) on the developing mouse brain, specifically during the perinatal period. The authors use high-resolution MRI to examine structural brain changes in neonates (postnatal day 7, P7) and compare these changes to those observed in adulthood. A key aspect of the study is the investigation of maternal care as a potential mediating factor in the effects of perinatal EE on neonatal brain development.

The work exhibits the following notable strengths:

(1) The study addresses a significant gap in the literature by investigating the effects of perinatal EE on whole-brain structure in neonates. Previous research has primarily focused on the effects of EE on the adult brain or specific aspects of early development, such as the visual system.

(2) The authors employ a combination of high-resolution MRI and behavioral analysis of maternal care, providing a comprehensive view of the effects of EE.

(3) The study reveals that EE affects brain structure as early as P7, with distinct regional changes compared to adulthood. The finding that maternal care influences neonatal brain structure and correlates with the effects of EE is particularly noteworthy.

(4) The paper is clearly written, well-organized, and easy to follow. The figures and tables are informative and effectively illustrate the key findings.

However, some weaknesses should be addressed to improve the quality of this study:

(1) While the study includes an assessment of maternal care, the observational period is relatively short. A more extended or continuous assessment of maternal behavior could provide a more comprehensive understanding of its role in mediating the effects of EE.

(2) The study primarily focuses on structural brain changes. Investigating the functional consequences of these changes could provide further insights into the long-term impact of perinatal EE.

(3) The study demonstrates a correlation between maternal care and neonatal brain structure but does not elucidate the underlying mechanisms. Future studies could explore potential molecular or cellular mechanisms involved in these effects.

Reviewer #2 (Public review):

This paper by Kaller and colleagues combines an interesting replication of findings on the importance of maternal behavior on brain development in the offspring with a state-of-the-art MRI analysis and a novel comparison between such perinatal and early postnatal enrichment via the activity of the mother and a classical enriched environment in the adult. In general, the observations are as one would have expected. Early postnatal enrichment and adult enrichment have differential effects, which is plausible because, as the source of these changes is environmental, and environmental means very different things at these different stages. The three data sets presented are really interesting, and while the comparison between them might not always be as straightforward as it seems, the cross-sectional phenotyping with MRI already provides very important material and allows for interesting insight. Most interesting is possibly the massive effect of housing conditions at P7.

In particular, the role of individual behavior differs. The authors highlight this role of the interaction with the environment, rather than the environment alone. Maternal care is a process that involves the pup.

Importantly, the study shows that being born into an enriched environment predates certain changes that are still available after exposure at a later stage, but that there are also important differences. Detailed interpretation of these effects is not easy, however.

Notably, the study does not include a condition of enrichment from birth into adulthood, and no analysis of the perinatal enrichment effects at an adult age. The timeline can be guessed from Figure 1b, but the authors might in places be more explicit about the fact that, indirectly and sometimes directly, animals of different ages (young adult versus adult) are compared. There is obviously no experience of maternal care in adulthood and no active exploration, etc in childhood. In part, this is what this paper is about, but it requires some thought for the reader to separate the more trivial from the more profound conclusions. Some more guidance would probably be welcome here. In general, Figure 4 is a great idea (and visually very appealing), but the content is not quite clear. "Adults born in EE vs. switched to EE in adulthood": this has, as far as I can tell, not been studied. What is compared are EE effects at two different time-points with two supposedly different mechanisms.

From such a more mechanistic side, the authors might, for example, want to relate the observed patterns to what is known about the developmental (and plastic) dynamics in the respective brain regions at the given time. But age is a confounder here.

There is another interesting point that the authors might discuss more prominently. The inter-individual differences in Z-score are dramatic within essentially all groups. So while the mean effects might still be statistically different, a large proportion of animals are within a range of values that could be found in either experimental group. The same is also true for the effects of maternal care, as depicted in Figure 3. While there is, for this ROI, a clear trend that overall relative volume decreases with maternal contact time at each time point, there is a large range of values for each maternal contact time bin. Consequently, neither genetics nor maternal care per se can be the driver of this variation. Part of it will be technical, but the trend in the data indicates that certainly not all of this is noise and technical error.

This study has some open ends but also provides a very important and interesting direction for future study, corroborating the idea that behavior, maternal and own, does matter.

Reviewer #3 (Public review):

Summary:

This study aimed to investigate the effect of environmental enrichment (EE) during the critical perinatal period on the developing brain structure and compare it with other periods. Different datasets of mice with EE or standard housing (SH) were compared with post-mortem MRI: dataset A (MRI at P96; 13 animals in EE during adulthood P53-P96, 14 animals in SH), dataset P (MRI at P43; 24 animals in EE during perinatal period and adulthood E17-P43, 25 animals in SH) and dataset N (MRI at P7; 52 animals in EE during perinatal period E13-P7, 67 animals in SH / resulting from 5 dams with 2 litters: 4 dams in EE and 6 dams in SH). The study replicated the effects observed during adulthood (main neuroanatomical EE/SH difference in datasets A and P: increase in the hippocampus volume) but also showed that volumetric changes for some regions differ between datasets A and P, suggesting different mechanisms of brain responses to enrichment depending on the period when EE was applied. Results on dataset N further showed that EE leads to lower brain size and differences for various regions: volume reduction in striatum, frontal, parietal, and occipital regions, hippocampus; volume increase for a few thalamic nuclei and hindbrain, suggesting different patterns of perinatal EE effects in datasets P and N. Since mice at P7 show little engagement with their environment, the authors further explored the hypothesis that the dams' behavior and interaction with neonates could be a mediator of brain differences observed at P7 between EE and SH animals. Maternal contact time was related to the P7 volumes for some regions (striatum, brainstem), but the variability and low sample size prevented a clear separation between EE and SH in terms of maternal behaviors.

Strengths:

(1) The question raised by this article is important at a fundamental level for our understanding of the complex interactions between the brain, behavior, and the environment.

(2) This study replicates previous observations on the effects of EE in adult mice.

(3) While some studies have been performed on neonates of dams exposed to EE during gestation, it is the first time that the effects of perinatal EE are investigated, in both the developing and mature brains with MRI. From a translational perspective, this is crucial for our understanding of human neurodevelopment in interaction with the environment.

(4) The analyses carried out are numerous and detailed.

Weaknesses:

(1) The analyses carried out do not allow us to fully assess whether differences in maternal care mediate the effects of EE on brain structure during development. The observations support this causal hypothesis, but a complete mediation analysis would be useful if permitted by the sample size and the variability observed between litters.

(2) The article is quite dense to read, given the number of analyses carried out. It is difficult at first reading to get a global view of the results. Figure 4 could be highlighted earlier to present the hypotheses and tests carried out.

(3) The figures could be more explicit in terms of legends (particularly the supplementary figures).