A reverse-phase HPLC and fluorescence detection method for measurement of 5-hydroxytryptamine (serotonin) in Planaria

Introduction:Planaria have proven to be a good model system in which to investigate mammalian behaviors and responses to drugs. We have recently studied the response of planarians to dopaminergic ligands and to the effects of cocaine and opioids. To correlate behavior (specifically, drug withdrawal) with neurotransmitter levels, we developed a method to quantify 5-hydroxytryptamine (5-HT; serotonin) in planarians. Methods: Following the homogenization of planarians in aqueous solvent (perchloric acid, l-cystine, and Na2EDTA) and centrifugation of supernatant (14,000×g at 4 °C for 20 min), 5-HT was measured using HPLC (aqueous citric acid buffer mobile phase; 5-μm C18 column with fluorescence detection, 280/340 nm). Nω-methyl-5-HT was used as an internal standard (IS). Results: 5-HT was rapidly extracted and conveniently measured from the planarians. The detection limit of the procedure (0.35 ng) was below the amount of 5-HT in one animal. Discussion: The ability to measure neurotransmitter levels provides a methodological opportunity to correlate behavior with biochemical changes in planarians and to extend behavioral observations to intracellular transmitter and second messenger transduction pathways.