Anxiety disorders are a significant public health concern, and physical exercise has emerged as an effective preventative and therapeutic strategy. Meta-analyses and animal studies support exercise's anxiolytic effects, alongside improvements in cognition following chronic stress. Proposed neurobiological mechanisms include enhanced neurogenesis, modulation of cytokines, and cerebrovascular changes. At the cellular level, exercise impacts neuronal, astrocytic, microglial, and oligodendrocytic morphology and function. However, the complete molecular signaling pathway remains unclear. The mTOR pathway, implicated in both chronic stress and exercise-induced effects, is a potential mediator. The medial prefrontal cortex (mPFC), crucial for mental functions and anxiety, is a focus of this study. The authors hypothesize that exercise may mediate the mTOR pathway in the mPFC to influence neural activity and alleviate anxiety. To test this, they employed a CRS model in adolescent mice, expecting to observe anxiety-like behaviors associated with reduced mPFC neuronal activity. They anticipate that exercise will prevent these phenotypes by enhancing mPFC neuronal activity through mTOR pathway activation, which protects axonal myelination.

Existing literature establishes the effectiveness of physical exercise in reducing anxiety symptoms across various populations and animal models. Studies have highlighted the positive impact of exercise on cognitive deficits caused by chronic stress, proposing several neurobiological mechanisms, including increased hippocampal neurogenesis, altered cytokine profiles, and cerebrovascular adjustments. At the cellular level, detailed investigations have shown the effects of exercise on various brain cell types. Although the cellular effects are well-documented, the complete molecular mechanisms, especially regarding signaling pathways, remain to be fully elucidated. The involvement of factors like BDNF in mediating the anxiolytic effects of exercise has been suggested, but a comprehensive picture is lacking. Previous research from the authors' group and others have demonstrated the role of the mTOR pathway in exercise-induced changes in motor cortex and its correlation with chronic stress responses. The mPFC's role in anxiety is also widely established, however in vivo studies exploring exercise's impact on mPFC activity and axonal myelination are limited.

This study utilized male C57BL/6J mice (4-6 weeks old). A chronic restraint stress (CRS) model was established by restraining mice for 3 hours daily for 14 consecutive days. A treadmill exercise group underwent 1-hour daily exercise for 14 days. Behavioral assays included the open field test, elevated plus maze test, and marble burying test. Molecular analyses involved RNA extraction and qPCR, protein extraction and Western blotting, immunofluorescent imaging, and stereotaxic injections. In vivo calcium imaging using two-photon microscopy was employed to monitor neuronal activity. Transmission electron microscopy (TEM) was used to assess axonal myelination. Statistical analysis included t-tests, ANOVA, and nonparametric tests as appropriate. Specific molecular manipulations included rapamycin treatment (mTOR inhibitor), AAV-mediated shRNA knockdown of Raptor (a core mTOR protein), and manipulation of Tsc2 (mTOR regulator). Chemogenetic approaches using DREADDs (designer receptors exclusively activated by designer drugs) were also employed to control neuronal activity.

The CRS model successfully induced anxiety-like behaviors without affecting general locomotor activity. Fourteen days of treadmill exercise effectively prevented these anxiety-related behaviors. Exercise also protected against CRS-induced axonal demyelination in the mPFC, as evidenced by increased MBP expression and improved g-ratio (axonal diameter/myelinated sheath diameter). Exercise increased the phosphorylation of mTOR, Akt, and ribosomal protein S6 in the mPFC. Pharmacological (rapamycin) and genetic (Raptor knockdown) inhibition of mTOR abolished the exercise-mediated anxiolytic effects and demyelination protection. In vivo calcium imaging showed that exercise preserved normal neuronal activity in the mPFC, which was also dependent on mTOR activation. Chemogenetic inhibition of mPFC neuronal activity blocked the exercise-induced protective effects on myelination and anxiety-like behaviors. Proteomic analysis revealed FMRP as a potential upstream regulator of mTOR; FMRP expression increased in the CRS group and decreased with exercise. Genetic knockdown of FMRP replicated the exercise-mediated effects. RNA methylation of the Fmr1 gene (encoding FMRP) was influenced by exercise. Interfering with RNA methylation using Alkbh5 overexpression or knockdown reversed the exercise-mediated effects. Supplementation with S-adenosyl methionine (SAM), a methyl donor, mimicked the effects of exercise on FMRP, mTOR, and anxiety-related behaviors.

The study demonstrates a clear link between exercise, mTOR pathway activation, and stress resilience in adolescent mice. The findings highlight the mPFC's crucial role in stress response and the activity-dependent nature of axonal myelination. The identification of an FMRP-mTOR pathway, regulated by exercise-mediated RNA methylation, provides a novel molecular mechanism underlying exercise's anxiolytic effects. These results are consistent with previous research showing the impact of environmental stressors on myelination, especially during developmental critical periods. The study also supports the idea that neuromodulation approaches could be used to manipulate white matter structure to treat mental disorders. The use of both pharmacological and genetic manipulations strengthens the conclusions, though the lack of cell-type specificity in the rapamycin studies is a limitation. The study expands our understanding of exercise's effects on white matter structure, complementing existing research showing beneficial effects in various neurological and psychiatric conditions.

This study provides strong evidence for an FMRP-mTOR pathway mediating the anxiolytic and myelin-protective effects of exercise in adolescent mice subjected to chronic stress. The findings illuminate the complex interplay between neuronal activity, myelination, and stress resilience at the molecular level. Future research could focus on translating these findings to human studies and exploring the potential therapeutic implications of targeting this pathway for treating anxiety and related disorders. Further investigation of the broader role of RNA methylation in brain plasticity is also warranted.

While the study used multiple methods to validate its findings, several limitations should be noted. The use of rapamycin, while effective, lacks cell-type specificity, potentially affecting mTOR activity beyond neurons. The study focused on male mice; future research should investigate sex differences. The specific metabolites mediating the exercise-induced changes in RNA methylation remain to be fully identified. The generalizability of the findings to other stress paradigms and age groups requires further study.