Mice lacking functional presynaptic active zone protein Bassoon are characterized by an enlarged cerebral cortex and an altered cortical activation pattern. This morphological and functional phenotype is associated with defined metabolic distortions as detected by a metabonomic approach using high-field (14.1 T) high-resolution 1H-nuclear magnetic resonance spectroscopy (MRS) in conjunction with statistical pattern recognition. Within the cortex but not in the cerebellum, concentrations of N-acetyl aspartate, glutamine, and glutamate are significantly reduced, whereas the majority of all other detectable low molecular metabolites are unchanged. The reduction of the neuron-specific metabolite N-acetyl aspartate in the cortex coincides with a significant decrease in neuronal density in cortical layer V. Comparing the neuron with glia cell densities across the cortex reveals cortex layer-dependent alterations in the ratio between both cell types. Whereas the ratio shifts significantly toward neurons in the cortical input layers IV, the ratio is reversed in cortical layer V. Consequently, the previously observed altered neuronal activation pattern in the cortex is reflected not only in defined cytoarchitectural anomalies but also in metabolic disturbances in the glutamine-glutamate and N-acetyl aspartate metabolism.