A reverse-phase HPLC and fluorescence detection method for measurement of 5-hydroxytryptamine (serotonin) in Planaria.
BACKGROUND
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 Na(2)EDTA) and centrifugation of supernatant (14,000 x g at 4 degrees C for 20 min), 5-HT was measured using HPLC (aqueous citric acid buffer mobile phase; 5-microm C(18) column with fluorescence detection, 280/340 nm). N(omega)-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.


CONCLUSIONS
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.
Umeda
Sumiyo
S
Department of Pharmaceutical Sciences, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA 19140, USA.
Stagliano
Gregory W
GW
Borenstein
Michael R
MR
Raffa
Robert B
RB
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015394:Molecular Structure; D010932:Planarians / Q000737:chemistry*; D012015:Reference Standards; D012701:Serotonin / Q000032:analysis* / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
D012701:Serotonin