1ST DIRECT DEMONSTRATION OF PREFERENTIAL RELEASE OF CITRATE FROM ASTROCYTES USING [C-13]NMR SPECTROSCOPY OF CULTURED NEURONS AND ASTROCYTES

Primary cultures of cerebral cortical neurons or astrocytes or the two cell types together (co-cultures) were incubated with [1-C-13]glucose for 20 or 48 h. Subsequently, perchloric acid (PCA) extracts of the cells as well as redissolved lyophilyzed media were subjected to NMR spectroscopy in order to detect C-13-labeled amino acids (glutamine, glutamate, gamma-aminobutyrate (GABA)) and other metabolites (lactate, tricarboxylic acid cycle (TCA) constituents). NMR spectra of PCA extracts of neurons or co-cultures exhibited distinct peaks for glutamate and GABA whereas the PCA extracts of astrocytes and co-cultures showed peaks corresponding to glutamine and glutamate. This pattern is consistent with the neuronal location of the GABA synthesizing enzyme glutamate decarboxylase and the astrocytic localization of the glutamine synthesizing enzyme, glutamine synthetase. NMR spectra of the incubation media showed clearly that C-13-labeled citrate, alanine and glutamine were synthesized and released from astrocytes since only media from the astrocyte cultures or co-cultures of neurons and astrocytes contained these metabolites in detectable amounts. It may be concluded that astrocytes play an important role supplying neurons with precursors for biosynthesis of glutamate and GABA such as glutamine and TCA cycle constituents. Since among the latter only citrate could be found in significant amounts it may be hypothesized that this may be the quantitatively most important TCA constituent to be released from astrocytes and subsequently utilized by neurons.