High dietary phosphate intake induces anxiety in normal male mice

Background: Diet is increasingly recognized as an important risk factor for mental health. Inorganic phosphate (Pi) is currently used as a flavor enhancer or preservative at an unregulated amount in the western diet despite evidence that excessive dietary Pi intake associates with metabolic and cardiovascular disorders. The impact of high Pi on brain function remains poorly understood. This study aimed to evaluate the effects of chronic consumption of high dietary phosphate on behavior, neurovascular health, and neuroimmune populations, and cortical gene expression in key brain regions associated with emotional regulation. Methods: Adult C57BL/6 male mice were fed either a normal phosphate (NP) or high phosphate (HP) diet for 12 weeks. Behavioral assessments included the open field test (OFT) and fear conditioning. Histological analyses assessed neuronal densities and vascularization. Flow cytometry quantified brain-resident immune cell populations and microglia. Unbiased analysis of hippocampal gene expression was performed using RNA sequencing (RNA-Seq). Results: HP-fed mice exhibited increased anxiety-like behaviors compared to NP-fed controls, as indicated by increased thigmotaxis (i.e., more time close to the walls and, consequently, less time spent in the central area, HP: 164 ± 61 vs. NP: 215 ± 54 s, P = 0.03) in the OFT and increased time freezing regardless of stimulus type during fear conditioning. Neuronal density is significantly decreased in the hypothalamus of HP-fed mice (21.9 % ± 4.5 % vs. 16.4 ± 2.9 %, P = 0.02) but without concomitant differences in brain vascularization. Immunophenotyping showed that HP-diet significantly reduced TCRβ⁺ T cells and NK1.1⁺ NK cells (both P < 0.05), suggesting diet-induced alterations in neuroimmune homeostasis. RNA-Seq identified significant alterations in gene expression in the hippocampus, including upregulation of Neat1 and Stat3 and downregulation of Igf2, which are implicated in stress regulation, neurodegeneration, synaptic plasticity and immune system pathways. Conclusions: Collectively, this study highlights that habitual consumption of high dietary phosphate in mice may induce chronic anxiety, accompanied by significant changes in the neuronal and brain-resident immune populations. The data point to a potential link between dietary Pi, neuroinflammation, and the pathogenesis of anxiety and depression in otherwise healthy young male mice. Given the prevalence of phosphate additives in processed foods, these findings have important public health implications supporting the regulation of Pi in the food industry.