reviously. Primers for cDNA amplification of EAAC1, Citrin/AGC2 and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were selected according to the rat sequences available in GenBank, in a region with high homology to the human genes. Primer optimization, i.e. dimerization, self-priming and melting temperature, was carried out with Primer Express. Gene-specific primers were blasted against the GenBank to confirm their species and gene specificity; the melting curve was used to determine the specificity of each primer. All samples from different 16614540” experiments were analyzed in triplicate in two assays to verify assay reproducibility, and the mean values of each point were used for gene expression quantification. Analysis of the melting curve confirmed the specificity of the PCR products. When reverse transcription PCR analysis was needed, it was performed as previously described by using primers shown in Acknowledgments The authors thank Gerardo Galeazzi, Franco Pettinari and Carlo Alfredo Violet for their excellent technical support. Author Contributions Conceived and designed the experiments: SM PC VL S. Arcangeli AAN AG S. Amoroso. Performed the experiments: SM VL PC S. Arcangeli AAN AG. Analyzed the data: SM VL PC S. Arcangeli AAN AG S. Amoroso. Wrote the paper: SM PC VL S. Amoroso. 19 Mitochondrial NCX1/EAAC1 Sustain Brain Metabolism References 1. Vanzetta I, Grinvald A Increased cortical oxidative metabolism due to sensory stimulation: implications for ” functional brain imaging. Science 286: 15551558. Sibson NR, Dhankhar A, Mason GF, Rothman DL, Behar KL, et al. Stoichiometric coupling of brain glucose metabolism and glutamatergic MS 275 neuronal activity. Proc Natl Acad Sci U S A 95: 316321. Pellerin L How astrocytes feed hungry neurons. Mol Neurobiol 32: 5972. Mangia S, Garreffa G, Bianciardi M, Giove F, Di Salle F, et al. The aerobic brain: lactate decrease at the onset of neural activity. Neuroscience 118: 710. Chih CP, Roberts Jr. EL Energy substrates for neurons during neural activity: a critical review of the astrocyte-neuron lactate shuttle hypothesis. J Cereb Blood Flow Metab 23: 12631281. Figley CR Lactate transport and metabolism in the human brain: implications for the astrocyte-neuron lactate shuttle hypothesis. J Neurosci 31: 47684770. Magistretti PJ, Pellerin L Astrocytes Couple Synaptic Activity to Glucose Utilization in the Brain. News Physiol Sci 14: 177182. McKenna MC, Waagepetersen HS, Schousboe A, Sonnewald U Neuronal and astrocytic shuttle mechanisms for cytosolic-mitochondrial transfer of reducing equivalents: current evidence and pharmacological tools. Biochem Pharmacol 71: 399407. Nehlig A, Coles JA Cellular pathways of energy metabolism in the brain: is glucose used by neurons or astrocytes Glia 55: 12381250. Panov A, Schonfeld P, Dikalov S, Hemendinger R, Bonkovsky HL, et al. The neuromediator glutamate, through specific substrate interactions, enhances mitochondrial ATP 
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