Treadmill exercise modulates the medial prefrontal-amygdala neural circuit to improve the resilience against chronic restraint stress

Physical exercise is an effective way to reduce the risk of anxiety disorders, supported by human and rodent studies. While research has focused on hippocampal neurogenesis, oxidative stress relief, and neuroinflammation as mechanisms, neural circuitry mechanisms remain unclear. Brain imaging studies in humans have shown altered connectivity in anxiety-related brain regions, including the amygdala, a crucial region for anxiety behaviors significantly affected by exercise. The basolateral amygdala (BLA) is a central hub receiving inputs from cortical and subcortical nuclei, integrating sensory and mental status. Chronic restraint stress (CRS) disrupts BLA synaptic plasticity and membrane conductance, leading to anxiety. The medial prefrontal cortex (mPFC)-BLA pathway, highly interconnected and activated in mouse CRS models, is a potential target. Given that exercise training alters connectivity between mPFC and amygdala in humans, this study hypothesizes that the mPFC-BLA circuit plays a role in exercise-improved resilience to environmental stress.

Existing literature extensively documents the anxiolytic effects of exercise in both human and animal models. Meta-analyses demonstrate the efficacy of exercise in treating anxiety and stress-related disorders. Studies have explored the mechanisms, primarily focusing on the impact of exercise on hippocampal neurogenesis, a process that generates new neurons in the hippocampus, a brain region involved in memory and mood regulation. Other proposed mechanisms include the reduction of oxidative stress and neuroinflammation in the brain. However, the precise neural circuitry mechanisms underlying these effects remain largely unexplored. While some studies have implicated altered connectivity within anxiety-related brain networks, a detailed understanding of the specific circuits involved and how exercise modulates them is lacking. This gap in knowledge motivated the current study to investigate the role of the mPFC-BLA pathway in mediating the anxiolytic effects of exercise.

This study utilized a mouse model of chronic restraint stress (CRS) to investigate the effects of treadmill exercise on anxiety-like behaviors and the underlying neural circuitry mechanisms. Adult male C57BL/6J mice were randomly assigned to four groups: control (Con), control with exercise (Con+Ex), CRS, and CRS with exercise (CRS+Ex). The CRS group underwent 3 hours of daily restraint stress for 14 consecutive days, while the exercise groups received 1 hour of daily treadmill exercise (10 m/min) after the restraint stress paradigm. Behavioral assays, including the open field test and elevated plus maze, were conducted to assess anxiety-like behaviors. To investigate the mPFC-BLA circuit, researchers employed several techniques: Retrograde tracing using adeno-associated virus (AAV) vectors was used to identify BLA-projecting mPFC neurons, enabling targeted electrophysiological recordings. Ex vivo electrophysiological recordings were conducted on acute brain slices to assess the excitability of these neurons and their excitatory-inhibitory (E/I) balance using whole-cell patch-clamp recordings, examining miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Chemogenetic techniques, utilizing AAV vectors expressing inhibitory (hM4Di) or excitatory (hM3Dq) DREADDs (designer receptors exclusively activated by designer drugs) in BLA-projecting mPFC neurons, were employed to manipulate circuit activity and observe the resulting behavioral changes. Anterograde transsynaptic tracing was used to identify and characterize the downstream BLA neurons innervated by the mPFC. Immunofluorescent staining was used to characterize the neuronal subtypes involved in the mPFC-BLA pathway. Statistical analyses, including ANOVAs and t-tests, were used to compare group differences.

The study found that 14 days of treadmill exercise effectively prevented the development of anxiety-like behaviors in mice subjected to chronic restraint stress. Electrophysiological recordings revealed that CRS resulted in hyperexcitation of the mPFC-BLA circuit, evident in increased excitability of both presynaptic mPFC neurons projecting to the BLA and postsynaptic BLA neurons. Exercise reversed this hyperexcitation, restoring neuronal activity to baseline levels. Chemogenetic inhibition of the mPFC-BLA pathway mimicked the anxiolytic effects of exercise, while chemogenetic activation of this pathway in exercised mice resulted in a relapse of anxiety-like behaviors, strongly supporting the causal role of this circuit in mediating exercise's effects. Further analysis showed that the majority of BLA-projecting mPFC neurons are glutamatergic and belong to the intratelencephalic (IT) subtype, rather than pyramidal tract (PT) neurons. Exercise training selectively inhibited BLA-projecting neurons in the mPFC while potentiating the activity of non-BLA projecting neurons, indicating a specific effect on anxiety-related pathways. Finally, the study demonstrated that exercise restored the E/I balance in both mPFC and BLA neurons, mitigating the imbalance induced by CRS.

These findings provide compelling evidence for a neural circuitry mechanism by which exercise improves resilience against stress-induced anxiety. The study directly demonstrates the causal involvement of the mPFC-BLA pathway in mediating the anxiolytic effects of exercise. The selective modulation of BLA-projecting mPFC neurons, distinct from other mPFC projections, highlights the complexity of cortical circuits in emotional processing and suggests that the overall mPFC activity should not be oversimplified when assessing anxiety or depression. The study's findings add to the existing literature by demonstrating a specific neural circuit mechanism underlying the beneficial effects of exercise on mental health, moving beyond the previously emphasized roles of neurogenesis, oxidative stress, and neuroinflammation. The results could guide future research into targeted interventions for anxiety disorders that focus on modulating specific brain circuits rather than global brain function.

This study demonstrates that 14 days of treadmill exercise effectively reduces anxiety-like behaviors in a mouse model of chronic restraint stress by attenuating the hyperexcitation of the mPFC-BLA neural circuit. The findings highlight the causal role of this circuit in mediating exercise's anxiolytic effects and suggest a novel target for therapeutic interventions. Future research should explore the specific molecular mechanisms underlying the exercise-induced modulation of this circuit, including potential peripheral factors such as blood-borne metabolites.

The study primarily used a mouse model, which may limit the generalizability of the findings to humans. While the chemogenetic approach provides strong evidence for the causal role of the mPFC-BLA circuit, it is a reductionist approach, and the complex interplay of other brain regions in regulating anxiety could not be fully elucidated within this study. Future work should examine this circuit's modulation in other stress paradigms and translate the findings to human studies. Further research is needed to understand the long-term effects of exercise and potential for relapse after exercise cessation.