Brief communication Measurement of glutamate and aspartate in Planaria

AbstractIntroduction: The major excitatory neurotransmitters in the mammalian central nervous system are glutamate and aspartate. We developed arapid and efficient method for the extraction and measurement of these amino acids in Planaria—a valuable model for mammalian processesbecause of their simple, centralized nervous system and similar neurotransmitter systems. Method: The method utilized buffer extraction(perchloric acid containing 0.025% of l-cystine and Na 2 EDTA), simple derivatization, high-pressure liquid chromatography (HPLC), andfluorescence detection. Results: The meanTS.E. M. amounts of glutamate and aspartate were 322.6T43. 6 and 188.6T27. 6 pmol/mg-planarian,respectively. Discussion: The method provides the ability to investigate changes in glutamate and aspartate in response to drug administration orwithdrawal.D 2005 Elsevier Inc. All rights reserved. Keywords: Aspartate; Glutamate; Excitatory amino acids; HPLC; Planaria 1. IntroductionGlutamate and aspartate are the major excitatory neuro-transmitters in the mammalian central nervous system. Thepresence of these amino acids at more than half of all CNSsynapses underscores their key involvement in learning,memory, movement disorders, drug addiction, and othernormal and abnormal physiological processes and behaviors.Planaria, a type of flatworm having bilateral symmetricalnerve processes consisting of cephalic ganglia and peripheralnerve cords (Sarnat & Netsky, 1985), provide a useful andconvenient model for the study of nervous system structure andfunction (Agata, 2002; Agata & Watanabe, 1999; Algeri et al.,1983; Carolei, Margotta, & Palladini, 1975; Newmark,Riedden, Cebria, & Sanchez Alvarado, 2003; Newmark S Palladini et al., 1996; Passarelli et al.,1999; Venturini et al., 1989). Our laboratory has developed andutilized a metric that quantifies withdrawal behavior inplanarians following exposure to cocaine and other drugs(Raffa & Desai, 2005; Raffa, Holland, & Schulingkamp, 2001;Raffa & Martley, 2005; Raffa & Valdez, 2001; Raffa,Stagliano, & Umeda, 2003; Raffa, Valdez, Holland, S Umeda, Stagliano, Borenstein, & Raffa,2005; Umeda, Stagliano, & Raffa, 2004). The withdrawal ofmammals from repeated exposure to psychoactive drugsinvolves excitatory amino acid transmitters.Previous studies have demonstrated that planarians containopioid peptides, 5-HT, catecholamines, and GABA (e.g.,Gustafsson, 1985; Umeda et al., 2005; Venturini, Carolie,Paladini, Margotta, & Lauro, 1983; Welsh & Williams, 1970). These results indicate that planarians synthesize and utilize anumber of the same transmitters that regulate neural processesin mammals. Because glutamate is the lone precursor forGABA in the mammalian brain (Roberts & Frankel, 1950), thepresence of GABA in Planaria suggests the existence ofglutamate. Indeed, anatomical and pharmacological evidencesuggests a functional role for glutamate in planarians,glutamate-like immunoreactivity was detected in anotherflatworm (Webb & Eklove, 1989), and a recent in situhybridization study reported a putative homolog of glutamatereceptors in Planaria (Cebria et al., 2002). An assay thatconveniently measures the change in glutamate and aspartatecontent in planarians would provide the opportunity tocorrelate withdrawal behaviors with alterations in glutamateor aspartate levels. In the present study, we developed aneurochemical method that rapidly measures glutamate andaspartate in planarians. The method utilizes a straightforwardextraction, simple derivatization, high-pressure liquid chroma-tography (HPLC), and fluorescence detection. To our knowl-