2'-Fucosyllactose (2'-FL) represents a significant constituent within human milk oligosaccharides, and its benefits for intestinal health and neurodevelopment in infants and young children have been widely recognized. However, the mechanism by which 2'-FL influences early-life behavioral development through the gut-brain axis remains incompletely understood. In this study, we investigated the effects of neonatal 2'-FL supplementation on behavioral development in Sprague-Dawley (SD) rats and explored its potential microbiota-metabolite regulatory mechanisms. After 4 consecutive weeks of oral gavage beginning on postnatal day 2, rats receiving 2'-FL showed significantly increased average movement speed and central-area exploration time in the open field test. From a systemic perspective, 2'-FL lowered blood diamine oxidase (DAO) and D-lactic acid (D-LA) concentrations while elevating colonic mRNA levels of tight junction protein-related genes, consequently enhancing intestinal barrier stability. In addition, 16S rRNA sequencing showed that 2'-FL reshaped the gut microbiota structure and significantly enriched Lactobacillus, Bacteroides, and short-chain fatty acid (SCFA)-associated taxa represented by Lachnospiraceae_NK4A136_group. Untargeted metabolomics revealed that 2'-FL induced systemic metabolic remodeling, characterized by upregulation of the bioactive metabolites dehydroepiandrosterone sulfate (DHEA-S) and deoxycholic acid (DCA), and downregulation of the glucocorticoid-related corticosterone. Western blot validation further showed higher hippocampal protein expression of postsynaptic density protein 95 (PSD95), brain-derived neurotrophic factor (BDNF), and synaptophysin in the 2'-FL group, supporting enhanced synaptic plasticity-related signaling. In summary, neonatal 2'-FL intervention enhanced exploratory behavior and was accompanied by increased hippocampal synaptic plasticity-related protein expression in rats, potentially through coordinated regulation of gut microbiota composition and systemic metabolism.
