A central question in manganese neurotoxicity concerns the focal neuronal damage in the globus pallidus. In the present study, we investigated specific pathways of [1-13C]glucose as well as of [2-13C]acetate in this brain region and the frontal cortex following 4-day manganese treatment by high-resolution NMR spectroscopy. Following administration of 50 mg/kg/day manganese, glutamine concentration in the globus pallidus was decreased to 67% of control values but increased in frontal cortex by 56%. Manganese treatment also caused pronounced changes in glutamine-glutamate-GABA interconversion in which region-selective differences were observed in the isotopomer pattern of GABA compared with that of glutamine when including the astrocyte-specific substrate [2-13C]acetate. In particular, decreased 13C-labeled glutamine, synthesized from [1-13C]glucose, paralleled accumulation of 13C-labeled GABA in globus pallidus but not in frontal cortex. On the other hand, increased synthesis of glutamine from [2-13C]acetate showed that GABA accumulation was not due to increased synthesis from astrocytic glutamine. Furthermore, treatment with manganese resulted in a selective decrease in N-acetyl-aspartate in the globus pallidus. These data illustrate the potential importance of alterations in neuronal metabolic function. In particular, neuronal metabolic derangements and regional differences in the ability of astrocytes to fulfill their contribution to the glutamine-glutamate-GABA cycle during the early phase of manganese neurotoxicity may be crucial in determining the severity of cellular injury. © 2007 Wiley-Liss, Inc.