THE ANALYSIS OF AMINO ACID NEUROTRANSMITTERS IN C. ELEGANS USING MICROCHIP ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION
Vincent Fiorentino, Nathan Oborny.
The University of Kansas, Lawrence, KS.
C. elegans is used to study various biological phenomena. Having had its entire genome sequenced allows for genetic manipulation enabling studies of numerous biological characteristics. Glutamic acid decarboxylase (GAD) converts glutamate to the neurotransmitter gamma-aminobutyric acid (GABA). The single gene encoding GAD is called unc-25 in C. elegans. Previous work suggests that mutations in unc-25 ablate the worm's ability to make GABA. The goal of this work is to validate that the unc-25 animals are entirely GABA free. Should GABA be detected, there would be evidence to support the existence of unknown alternative pathways for GABA production. Understanding the role and pathways for GABA in C. elegans is important in understanding its nervous system which mirrors higher organisms such as humans. Because GABA exists in such low concentrations, preliminary work has focused on other amino acid neurotransmitters that exist at higher concentrations: glutamate, aspartate, and glycine. By fluorescently tagging the desired amino acid neurotransmitters and using microchip electrophoresis (ME) with laser induced fluorescence detection (ME-LIF), one can separate and selectively detect with high sensitivity each targeted analyte. Starting with these 3 amino acids has permitted verification of the extraction method as well as the ME-LIF separation-based assay vis-a-vis their ratio of charge to hydrodynamic radius. Additionally the small channel size offered with ME allowed for the analysis of nanoliter-sized samples.