BACKGROUND: Synapses can adapt to changes in the intracerebral microenvironment by regulation of presynaptic neurotransmitter release and postsynaptic neurotransmitter receptor expression following hypoxic ischemia (HI) injury. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) exerts a protective effect on neurons after HI and may be involved in maintaining the function of synaptic networks. In this study, we investigated the changes in the expression of NAAG, glutamic acid (Glu) and metabotropic glutamate receptors (mGluRs), as well as the dynamic regulation of neurotransmitters in the brain after HI, and assessed their effects on synaptic plasticity of the cerebral cortex. METHODS: Thirty-six Yorkshire newborn pigs (3-day-old, males, 1.0-1.5 kg) were selected and randomly divided into normal saline (NS) group (n = 18) and glutamate carboxypeptidase II inhibition group (n = 18), both groups were divided into control group, 0-6 h, 6-12 h, 12-24 h, 24-48 h and 48-72 h groups (all n = 3) according to different post-HI time. The content of Glu and NAAG after HI injury were detected by 1H-MRS scanning, immunofluorescence staining of mGluRs, synaptophysin (syph) along with postsynaptic density protein-95 (PSD95) and transmission electron microscopy were performed. ANOVA, Tukey and LSD test were used to compare the differences in metabolite and protein expression levels among subgroups. Correlation analysis was performed using Pearson analysis with a significance level of α = 0.05. RESULTS: We observed that the NAAG and mGluR3 expression levels in the brain increased and then decreased after HI and was significantly higher in the 12-24 h (P < 0.05, Tukey test). There was a significant positive correlation between Glu content and the expression of mGluR1/mGluR5 after HI with r = 0.521 (P = 0.027) and r = 0.477 (P = 0.045), respectively. NAAG content was significantly and positively correlated with the level of mGluR3 expression (r = 0.472, P = 0.048). When hydrolysis of NAAG was inhibited, the expression of synaptic protein PSD95 and syph decreased significantly. CONCLUSIONS: After 12-24 h of HI injury, there was a one-time elevation in NAAG levels, which was consistent with the corresponding mGluR3 receptor expression trend; the NAAG maintains cortical synaptic plasticity and neurotransmitter homeostasis by inhibiting presynaptic glutamate vesicle release, regulating postsynaptic density proteins and postsynaptic receptor expression after pathway activation. Video abstract.