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Abstract

The extracellular fluid (ECF) is the complex, dynamic environment embedding the neurons where neurochemical communication and metabolite transport occurs. Monitoring the composition of the fluid will lead to important insights into the understanding neuronal communication and function. We obtained ECF samples from the monkey cortex using a direct sampling technique. Chemical analysis of the six neurotransmitters acetylcholine, serotonin, dopamine, GABA, glutamate and aspartate was performed by hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS). All of the six compounds were eluted out within 11 min as baseline separation on a capillary HILIC column. The detection limits for acetylcholine, serotonin, dopamine, GABA, glutamate and aspartate were 0.06, 1.56, 0.63, 1.56, 6.25 and 156.25 femtomol, respectively. Extracellular fluid was withdrawn at 10-50 nl/min by direct sampling method and collected in vials for sequence analyses.
We have successfully obtained and analyzed ECF samples from the primary visual (V1), extrastriate visual (V4) and prefrontal (PF) cortex from awake as well as anesthetized animals. We have detected significant quantities of GABA, glutamate, dopamine, acetylcholine and aspartate. Preliminary findings indicate that both the brain region as well as the animal’s state influence neurotransmitter concentrations. Thus, we were able to detect dopamine in the prefrontal but not in visual cortex, whereas significant quantities of glutamate and GABA were present in both regions.
HILIC-MS/MS represents a powerful and robust technique for simultaneous quantification of neurotransmitters from ECF samples.