Chronic Unpredictable Mild Stress Induces Anxiety-Like Behavior, Ventral Tegmental Area Proteomic Alterations, and Systemic Metabolic Changes in Wistar Rats

Background: Chronic stress is a major risk factor for psychiatric disorders, including anxiety, and induces widespread neurobiological alterations. However, its impact on the mesolimbic reward system, particularly within the ventral tegmental area (VTA), a central regulator of motivation and affective behavior, remains incompletely understood. Here, we investigated whether chronic unpredictable mild stress (CUMS) induces coordinated peripheral metabolic alterations that are associated with proteomic changes in the VTA and behavioral outcomes. Methods: Male Wistar rats (n=56) were exposed to CUMS for two weeks, followed by behavioral testing, ^1H nuclear magnetic resonance–based plasma metabolomics, plasma cytokine quantification, shotgun proteomic analysis of the VTA, and cecal short-chain fatty acid profiling. Results: Chronically stressed animals exhibited anxiety-related behavior without evidence of anhedonia. Plasma metabolomic analysis revealed reduced myo-inositol levels and increased concentrations of glycine, uridine, and butyrate. In parallel, plasma interferon γ levels were significantly decreased following stress exposure. Proteomic profiling of the VTA revealed alterations in proteins involved in actin filament binding, cytoskeletal organization, and myo-inositol metabolism, suggesting stress-induced remodeling of synaptic architecture and intracellular signaling pathways. No significant differences were observed in cecal short-chain fatty acid content. Conclusion: Together, these findings indicate that chronic stress elicits anxiety-like behavior accompanied by systemic metabolic and immune alterations that converge with structural and metabolic changes in the VTA. These results highlight myo-inositol–related pathways as a potential link between peripheral metabolic dysregulation and stress-induced neurobiological vulnerability.