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A growing body of evidence suggests glutamate excess in schizophrenia and that N-methyl-d-aspartate receptor (NMDAR) hypofunction on γ-aminobutyric acid (GABA) interneurons disinhibiting pyramidal cells may be relevant to this hyperglutamatergic state. To better understand how NMDAR hypofunction affects the brain, we used magnetic resonance spectroscopy and resting-state functional magnetic resonance imaging (MRI) to study the effects of ketamine on hippocampal neurometabolite levels and functional connectivity in 15 healthy human subjects. We observed a ketamine-induced increase in hippocampal Glx (glutamate+ glutamine; F= 3.76; P= 0.04), a decrease in fronto-temporal (t= 4.92, P FDR< 0.05, k E= 2198, x=− 30, y= 52, z= 14) and temporo-parietal functional connectivity (t= 5.07, P FDR< 0.05, k E= 6094, x=− 28, y=− 36, z=− 2), and a possible link between connectivity changes and elevated Glx …