Rat brain primary neurons immobilized in basement membrane gel threads: an improved method for on-line 13C NMR spectroscopy of live cells.
In vivo MRS studies on intact brain reflect the metabolism of all cells present, but do not distinguish between different cell types. NMR studies of immobilized cultured primary cells, such as neurons and astrocytes, are a useful model to monitor the specific differences in metabolism of the various cell types in the brain. The present study shows that primary rat neuronal cells can be cultured in basement membrane gel threads. After 4 days of incubation the threads are filled with viable cells, and represent a population of morphologically differentiated neuronal cells with less than 5% of non-neuronal cells, i.e., astrocytes. These threads were placed into a NMR tube and used for on-line monitoring of neuronal metabolism. Under these conditions cells remained viable and metabolically active for several days. The energy status was monitored by using 31P NMR spectroscopy. To study neuronal glucose metabolism [1-13C]glucose was added to the perfusion medium and 30 min later 13C-labeled metabolites were detectable by 13C NMR spectroscopy. Immobilized neurons synthesized glycolytic products such as [3-13C]lactate and [3-13C]alanine, as well as several tricarboxylic acid cycle products, i.e., [2-13C]glutamate, [3-13C]glutamate, [4-13C]glutamate, [2-13C]aspartate, and [3-13C]aspartate. In summary, 31P and 13C NMR spectra can be recorded from live neuronal cells for up to 24 h using the newly designed procedure described in the present communication.
Brand
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A
Department of Chemistry, University of Bremen, D-28334, Bremen, Germany. brand@chemie.uni-bremen.de
Richter-Landsberg
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C
Flögel
U
U
Willker
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W
Leibfritz
D
D
D000818:Animals; D001485:Basement Membrane / Q000737:chemistry; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D015194:Diffusion Chambers, Culture / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D005782:Gels; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009474:Neurons / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D051381:Rats
D005782:Gels