Long-term intermittent fasting induces region- and sex-specific changes in astrocyte morphology and expression and anxiety-like behaviors in CD-1 mice

Intermittent fasting (IF) is a metabolic intervention that alters systemic and cerebral energy dynamics, promoting neurochemical and cellular adaptations that may influence mood and emotional regulation. Astrocytes, as key regulators of brain metabolism, neurotransmitter clearance, and neuroinflammatory processes, are particularly sensitive to fluctuations in nutrient availability. Dysregulation of astrocytic morphology and glial fibrillary acidic protein (GFAP) expression has been implicated in the pathophysiology of anxiety. However, the long-term effects of IF on astrocyte integrity and behavior remain poorly understood. This study examined how a six-week 16:8 IF protocol, initiated at postnatal day (PD) 60, modulates astrocytic morphology and GFAP expression in brain regions involved in emotional and homeostatic processing, including the medial prefrontal cortex (mPFC), insular cortex (IC), and amygdala (AM), in male and female CD1 mice. Anxiety-like behavior was assessed on PDs 102–103 using the Open Field Test (OFT), Elevated Plus Maze (EPM), and Light-Dark Test (LDT). Immunohistochemistry and Sholl analysis were used to quantify astrocyte number, cytoplasmic area, and branching complexity. IF increased anxiety-like behavior, particularly in the EPM. Region- and sex-specific astrocytic remodeling was observed: IF reduced cytoplasmic area and branching in the mPFC, induced modest changes in the IC, and produced robust increases in GFAP-positive astrocyte density and proximal arborization in the AM. Notably, anxiety indices positively correlated with astrocyte number in the AM, suggesting a potential link between glial reactivity and emotional responses to metabolic stress. These findings highlight the amygdala as a key site of astrocytic sensitivity to IF and underscore the importance of brain region and sex as modulators of glial adaptation.