Ketamine, a noncompetitive N-methyl-D-aspartate receptor antagonist, is associated with chronic abuse leading to schizophrenia-like cognitive deficits. The gut-brain axis may play a role in mediating substance-induced neurotoxicity; however, its involvement in ketamine-induced cognitive impairment remains poorly understood. Here, chronic ketamine exposure was administered intraperitoneally to C57BL/6N mice to examine its effects on gut microbiota homeostasis and associated amino acid metabolism. Cognitive deficits were evaluated using the Y-maze and novel object recognition (NOR) tests. Hippocampal ultrastructure was assessed by transmission electron microscopy (TEM). Multi-omics integration included 16S rRNA sequencing, untargeted and targeted plasma metabolomics, and Spearman correlation analysis. The results showed that ketamine-exposed mice exhibited significant cognitive impairments, including impaired spontaneous alternation in the Y-maze (P < 0.05) and a reduced discrimination index in the NOR test (P < 0.01). TEM analysis revealed hippocampal mitochondrial damage, accompanied by chromatin condensation. Gut microbiota analysis indicated dysbiosis, with a notable increase in Lachnospiraceae, Bacteroidaceae, Helicobacteraceae, and Rikenellaceae and a decrease in Verrucomicrobiaceae and Prevotellaceae at the family level. Plasma amino acid levels were also disrupted, with a significant decrease in L-glutamine, L-lysine, L-threonine and an increase in L-cysteic acid. Furthermore, strong correlations were observed between the abundance of Bacteroides, branched-chain amino acids (BCAAs), and cognitive scores (|ρ| > 0.6, P < 0.05). This study identifies the microbiota-amino acid-mitochondrial axis as the underlying mechanism driving ketamine-induced neurotoxicity. It highlights the correlation between gut microbiota-associated amino acid reprogramming and this process, offering potential targets for microbiome-based interventions to combat substance-related cognitive impairments.
