Intracisternal injection of [U-13C]glucose for investigating brain metabolism in freely moving mice

Mauro DiNuzzo, Simon Sanggaard, Serhii Kostrikov, Anna L. Xavier, Sofie Christensen, Blanca Aldana, Lasse K. Bak, Ursula Sonnewald, Arne Schousboe, Helle S. Waagepetersen, Maiken Nedergaard

PURPOSE: To investigate brain metabolism using intracisternal delivery of [U-13C]glucose, thus bypassing blood-brain barrier and avoiding effects of peripheral metabolism.

METHODS: Mice (C57BL/6JRj, 8wo) were implanted a chronic cannula into cisterna magna. After recovery (24h) an isosmolar 0.3M [U-13C]glucose solution was infused using a microinjection pump. Animals were sacrificed by microwave irradiation. 13C-labeling and metabolite amounts were determined using mass spectrometry and HPLC. Glycogen content was determined as glucose units after amyloglucosidase treatment.

RESULTS: [U-13C]Glucose injected at 2µL/min (10µL) resulted in fast label incorporation into brain lactate as well as glutamate and glutamine. Lactate labeling rapidly (within 10 min) decreased by about 50%, while enrichment in glutamate and glutamine kept increasing in the same time interval. Lactate was the only labeled compound recovered in cervical lymph nodes. [U-13C]Glucose injected at 0.3µL/min (4.5-18µL) resulted in progressive rise of label incorporation into brain lactate, glutamate, glutamine, aspartate and GABA. Labeling of these compounds was significantly faster in awake than anesthetized animals. The absolute concentrations of glutamate and GABA were higher in the awake state whereas that of glutamine was lower (~20% changes). Brain glycogen was higher (+50%) during anesthesia and was negatively correlated with glutamate/GABA and positively correlated with glutamine.

CONCLUSIONS: Our results indicate that lactate is produced in excess of its utilization and rapidly leaves the brain, possibly through brain lymphatics. The rate of aerobic glycolysis is higher during wakefulness than anesthesia and so is the rate of transmitter synthesis, suggesting higher glutamatergic and GABAergic tone in awake animals. The correlations between brain glycogen content and glutamate/GABA and glutamine in different states indicate that glycogen synthesis/breakdown is modulated by brain activity and contributes as substrate to neurotransmitter synthesis, underlining its functional importance.

Journal of Neurochemistry

DOI: 10.1111/jnc.14093

Green Open Access

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