Abstract
Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K+) release of preloaded [2,3-3H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K+) conditions from either [1,6-13C]glucose or [U-13C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 μM) or by replacing Ca2+ with Co2+. In the presence of both tiagabine and Co2+ (1 mM), release was abolished completely. The release observed in the presence of 25 μM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-13C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-13C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity.
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Abbreviations
- KR:
-
Krebs–Ringer
- TCA:
-
Tricarboxylic acid
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Acknowledgements
The expert secretarial assistance of Ms Hanne Danø and the skilful technical assistance of Ms Ann Lene Vigh are highly appreciated. The experimental work has been supported by grants from the Danish Medical Research Council (22-04-0314) and the Lundbeck, Hørslev and Novo Nordisk Foundations.
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Special issue article in honor of Dr. Ricardo Tapia.
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Waagepetersen, H.S., Døring, S. & Schousboe, A. Metabolism of [1,6-13C]Glucose and [U-13C]Glutamine and Depolarization Induced GABA Release in Superfused Mouse Cerebral Cortical Mini-slices. Neurochem Res 33, 1610–1617 (2008). https://doi.org/10.1007/s11064-008-9695-8
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DOI: https://doi.org/10.1007/s11064-008-9695-8