Quantitation of neurotransmitters and metabolites in microdialysate fluid and brain using advanced gas chromatographymass spectrometry (GC-MS)
Purpose: Sensitive and reliable measurement of neurotransmitters and other metabolites in brain is important for examining changes in brain biochemistry and metabolism. Our aim was to develop reliable analytical protocols for accurate measurement of different neurotransmitters and metabolites in cerebral spinal fluid (CSF) collected via microdialysis, focusing initially on several amino acids relevant for neurotransmission. The effect of potassium stimulation as a physiological control on extracellular levels of these compounds was also investigated. Method: CSF samples from the hippocampus and prefrontal cortex of male C57BL/6 mice (n=6 per region) were collected via microdialysis (flow rate 1 μl/min), before and after an infusion of artificial CSF containing 50 nM K+. Following heavy isotopic dilution, metabolite extraction and derivatisation, samples were analysed using a triple quadrupole GC-MS and quantitated using calibration standards. Results: 10 μl CSF dialysate was sufficient for accurate measurement of several neurotransmitters and several metabolites including GABA (mean 40 nM ±6 SEM), glutamate (45 nM ±4), alanine, dopamine, glycine and serine. Stimulation with 50 nM K+ induced a significant 2-3-fold increase of alanine, GABA and glycine (p<0.05). Conclusions: GC-MS targeted analysis provides a sensitive tool for measuring a large range of neurotransmitters and metabolites during a single analysis in small aliquots of CSF. Small amounts of brain, plasma and other tissues can also be analysed. We are currently monitoring metabolic changes in a transgenic R6/1 mouse model of Huntingtons disease to examine mechanisms of neurodegeneration and identify potential biomarkers.
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