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Zusammenfassung:
Changes in the concentration of neurochemicals, including neurotransmitters, neuromodulators and metabolites, reflect the complex interactions within local and global neural networks. The assessment of such changes promises thus insights into neural communications; in particular if several neurochemicals can be studied at the same time. Here we report findings from chemical analysis of six polar neurochemicals, including acetylcholine (ACh), choline, glutamine, glutamate, lactate and pyruvate, by means of hydrophilic interaction chromatography (HILIC) coupled to tandem mass spectrometry (MS/MS).
Extracellular fluid was withdrawn at 40 nl/min by nano push-pull perfusion (NFP3) and collected in vials for sequence analyses. We have successfully obtained and analyzed ECF samples from the primary visual (V1) and extrastriate visual (V4) cortex of awake or anesthetized non-human primates (macaca mulatta). The results showed that all six compounds can be determined by HILIC-MS with low sample treatment requirement. The sensitivity of our measuring system allows the simultaneous monitoring of these six compounds from a 200 nL in vivo sample. We further studied the effects of intracortical application of
cholinergic agents in the cortex on the concentration of these chemicals in the extracellular brain fluid. We observed significant increases in extracellular neurochemicals like glutamate and glutamine after the application of ACh or nicotinic agonists. During the ACh application experiments, the profile of exogenously applied ACh and its product choline were monitored, showing that ACh itself remained at low nM range 30 min after injection of 1 mM solution, due to the fast cholinergic metabolism in the brain. All in all, we conclude that capillary HILIC-MS combined with NFP3 is a sensitive technique for simultaneous monitoring of multi-class neurochemicals from extracellular brain samples. Future experiments will concentrate on the effects of the animal's internal state on the concentration of such neurochemicals.
