Rapid urbanization has reduced children's interaction with nature, potentially increasing their risk of stress and depression. While outdoor nature-related activities are known to enhance well-being, the underlying mechanisms remain unclear. This study aimed to investigate the impact of a structured nature-related program on the gut microbiota, fecal serotonin, and perceived stress in preschool children. Aggressive behavior in early childhood is a significant concern, often overlooked as a risk factor for future violence. The growing understanding of the brain-gut axis highlights the bidirectional influence of the gut microbiome on mental health. Previous research has demonstrated that stressors can alter gut microbiota composition and impact stress response and behavior. Serotonin, a neurologically active substance produced and modulated by the gut microbiome, is crucial in various physiological and psychological processes. While the link between gut microbiota, brain development, and behavior is established, studies involving exposure to natural outdoor environments are limited. Wilson's biophilia theory posits that natural environments are associated with positive health outcomes, including improved psychological well-being and reduced risk of psychiatric disorders. Increased exposure to natural environments influences the composition of the gut, skin, and salivary microbiomes. The 'Play&Grow' program, with its 'Connectedness to Nature' component, was designed to enhance biophilia and improve preschoolers' health. This trial specifically investigated the program's impact on the intestinal microbiome, gut serotonin levels, and the psychological well-being of 2-5-year-old children.

The literature review section supports the study's hypothesis by citing existing research on the impact of stress on gut microbiota, the role of serotonin in modulating behavior, and the benefits of nature exposure on mental well-being. Studies on animals and humans showed that stress is linked to changes in gut microbiota composition, particularly reduced *Lactobacilli* levels. The importance of the gut microbiome's role in producing neurologically active substances like GABA, which is linked to anxiety and depression, is highlighted. The review also cites studies indicating the potential contribution of short-chain fatty acids (SCFAs), metabolic byproducts of gut microbiota, to depression. While research exists on the connection between intestinal microbiota, brain development, and behavior, studies specifically focusing on the impact of outdoor nature exposure are limited. The review emphasizes Wilson's biophilia hypothesis and its association with positive health outcomes, decreased risk of psychiatric disorders, and improvements in psychological well-being. Studies examining the effect of exposure to natural environments and animals on gut, skin, and salivary microbiome composition are also referenced, supporting the rationale behind the intervention's design.

This study employed a two-arm, randomized controlled trial (RCT) with masked outcome assessment. Fifty-four families with preschool children (aged 2-5 years) were randomly assigned to either an intervention group (IG, n=30) or a control group (CG, n=24). Exclusion criteria included children from non-local families, children on antibiotics in the preceding two months, and children with chronic health conditions. The 10-week intervention (IG) involved weekly sessions in public parks, focusing on guided nature activities promoting hands-on experiences with natural materials. Families also received homework assignments to further increase their interaction with the outdoor environment. The control group (CG) received no intervention but were reminded of general healthy lifestyle recommendations. Pre- and post-intervention assessments included questionnaires measuring connectedness to nature (CN) and perceived stress (PSS-C), fecal serotonin level measurement via ELISA, and gut microbiota profiling using 16S rDNA amplicon sequencing. Data analysis involved comparing pre- and post-intervention changes in both groups, examining correlations between variables, and performing canonical correspondence analysis (CCA) to identify factors influencing gut microbiota composition. Statistical analyses included Wilcoxon signed-rank test, Jonckheere-Terpstra test, Spearman's rank correlation, logistic regression, repeated measures correlation coefficient, and CCA.

The 10-week intervention resulted in a significant increase in connectedness to nature (CN) in the IG (p=0.04). Fecal serotonin levels remained stable in the IG, while showing a decreasing trend in the CG (p=0.07). The IG showed significant improvements in overall PSS-C scores (p=0.05), anger frequency (p=0.01), and prosocial behavior (p=0.04) compared to the CG. A negative correlation was found between fecal serotonin and PSS-C score (r=-0.45, p=0.02), and a positive correlation with adequate sleep frequency (JT = 832, p<0.01). The gut microbiota analysis revealed changes in the IG, including a significant decrease in Bacteroidetes richness (p<0.01) and an increase in Proteobacteria richness (p=0.03). *Roseburia* abundance increased in the CG but not in the IG. Six species showed a positive correlation with fecal serotonin, and twelve showed a negative correlation. *Roseburia* species exhibited a significant negative correlation with fecal serotonin (p=0.01). The PSS-C score negatively correlated with Bacteroidetes diversity. Children with higher anger frequencies displayed less inter-individual variation in gut microbial communities and higher *Roseburia* levels. The intervention significantly altered microbiota functions in the IG, with a notable increase in Indole alkaloid biosynthesis (negatively correlated with PSS-C) and decrease in glycosphingolipid biosynthesis.

The findings support the hypothesis that nature-related activities positively influence preschool children's psychosocial well-being through alterations in gut microbiota and serotonin levels. The sustained fecal serotonin levels in the intervention group, in contrast to the control group's decrease, suggest a protective effect. The observed changes in gut microbiota composition, particularly the modulation of *Roseburia* abundance, are intriguing and warrant further investigation. The negative correlation between *Roseburia* and fecal serotonin aligns with previous findings linking *Roseburia* to mood disorders. The improvements in psychosocial scores align with systematic reviews showing the beneficial impact of nature exposure on negative emotions. The observed changes in gut microbiota functions, especially the increase in Indole alkaloid biosynthesis and decrease in glycosphingolipid biosynthesis, highlight potential pathways linking nature exposure, gut microbiome, and mental health. The results support the “Hygiene Hypothesis” suggesting that exposure to diverse microbes may be beneficial for immune system development and mental health.

This study provides pioneering evidence of the impact of nature-related activities on gut microbiota, fecal serotonin, and psychosocial behavior in preschool children. The Play&Grow intervention positively affected microbiota diversity, *Roseburia* abundance, fecal serotonin levels, and children's moods, particularly anger levels. Further research is needed to confirm the mechanistic links between connectedness to nature, gut microbiota, serotonin, and improved psychosocial behavior. Future studies should utilize larger sample sizes, metagenomic sequencing, and explore the diurnal fluctuations of fecal serotonin.

The study's limitations include the relatively small sample size, the duration of the intervention, and the limitations of the 16S rRNA reference database which hindered species-level resolution for some taxa. The study did not account for fecal sample collection time, potentially influencing serotonin level measurements. The lack of a detailed dietary assessment could have influenced the results as dietary factors could also impact the microbiome and serotonin levels. Despite these limitations, the findings provide valuable insights for future research.