There is a growing interest in biological models to investigate the effect of neurotransmitter dysregulation on the structure and function of the central nervous system (CNS) at different stages of development. Zebrafish, a vertebrate model increasingly used in neurobiology and neurotoxicology, shares the common neurotransmitter systems with mammals, including glutamate, GABA, glycine, dopamine, norepinephrine, epinephrine, serotonin, acetylcholine, and histamine. In this study, we have evaluated the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the multiresidue determination of neurotransmitters and related metabolites. In a first step, ionization conditions were tested in positive electrospray mode and optimum fragmentation patterns were determined to optimize two selected reaction monitoring (SRM) transitions. Chromatographic conditions were optimized considering the chemical structure and chromatographic behavior of the analyzed compounds. The best performance was obtained with a Synergy Polar-RP column, which allowed the separation of the 38 compounds in 30 min. In addition, the performance of LC-MS/MS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method was able to quantify 27 of these neurochemicals in zebrafish chemical models for mild (P1), moderate (P2), and severe (P3) acute organophosphorus poisoning (OPP). The results show a general depression of synaptic-related neurochemicals, including the excitatory and inhibitory amino acids, as well as altered phospholipid metabolism, with specific neurochemical profiles associated to the different grades of severity. These results confirmed that the developed analytical method is a new tool for neurotoxicology research using the zebrafish model.
Neurotransmitters LC-MS/MS Quality parameters Zebrafish larvae Animal models of human disease Acute organophosphorus poisoning
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The research leading to these results has received funding from the European Research Council under European Union's Seven Framework Programme (FP/2007e2013)/ERC Grant Agreement n.320737, the NATO SfP project MD.SFPP 984777, and the grant CTM 2014-51985 from the Spanish Ministry of Economy, Industry and Competitiveness. Moreover, Rita Bausano is acknowledged for extraction samples support during her Erasmus Traineeship stage in the Environmental Chemistry Department at IDEA-CSIC (Barcelona, Spain).
Compliance with Ethical Standards
Conflict of interest
The authors declare no financial conflict of interest.
Horzmann KA, Freeman JL. Zebrafish get connected: investigating neurotransmission targets and alterations in chemical toxicity. Toxics. 2016;4(3):19.CrossRefGoogle Scholar
Kurian MA, Gissen P, Smith M, Heales SJ, Clayton PT. The monoamine neurotransmitter disorders: an expanding range of neurological syndromes. Lancet Neurol. 2011;10(8):721–33.CrossRefGoogle Scholar
Ng J, Papandreou A, Heales SJ, Kurian MA. Monoamine neurotransmitter disorders – clinical advances and future perspectives. Nat Rev Neurol. 2015;11(10):567–84.CrossRefGoogle Scholar
Marecos C, Ng J, Kurian MA. What is new for monoamine neurotransmitter disorders? J Inherited Metab Dis. 2014;37(4):619–26.CrossRefGoogle Scholar
Pearl PL, Hartka TR, Taylor J. Diagnosis and treatment of neurotransmitter disorders. Curr Treat Opt Neurol. 2006;8(6):441–50.CrossRefGoogle Scholar
Pearl PL, Capp PK, Novotny EJ, Gibson KM. Inherited disorders of neurotransmitters in children and adults. Clin Biochem. 2005;38(12):1051–8.CrossRefGoogle Scholar
Babin PJ, Goizet C, Raldúa D. Zebrafish models of human motor neuron diseases: advantages and limitations. Prog Neurobiol. 2014;118:36–58.CrossRefGoogle Scholar
Faria M, Prats E, Padrós F, Soares AM, Raldúa D. Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication. Arch Toxicol. 2016:1–11.Google Scholar
Raldúa D, Piña B. In vivo zebrafish assays for analyzing drug toxicity. Expert Opin Drug Metab Toxicol. 2014;10(5):685–97.CrossRefGoogle Scholar
Tingaud-Sequeira A, Raldúa D, Lavie J, Mathieu G, Bordier M, Knoll-Gellida A, et al. Functional validation of ABHD12 mutations in the neurodegenerative disease PHARC. Neurobiol Dis. 2017;98:36–51.CrossRefGoogle Scholar
Rico E, Rosemberg D, Seibt K, Capiotti K, Da Silva R, Bonan C. Zebrafish neurotransmitter systems as potential pharmacological and toxicological targets. Neurotoxicol Teratol. 2011;33(6):608–17.CrossRefGoogle Scholar
Panula P, Sallinen V, Sundvik M, Kolehmainen J, Torkko V, Tiittula A, et al. Modulatory neurotransmitter systems and behavior: towards zebrafish models of neurodegenerative diseases. Zebrafish. 2006;3(2):235–47.CrossRefGoogle Scholar
Sallinen V, Sundvik M, Reenilä I, Peitsaro N, Khrustalyov D, Anichtchik O, et al. Hyperserotonergic phenotype after monoamine oxidase inhibition in larval zebrafish. J Neurochem. 2009;109(2):403–15.CrossRefGoogle Scholar
Tran S, Nowicki M, Muraleetharan A, Chatterjee D, Gerlai R. Neurochemical factors underlying individual differences in locomotor activity and anxiety-like behavioral responses in zebrafish. Prog Neuro-Psychopharmacol Biol Psychiat. 2016;65:25–33.CrossRefGoogle Scholar
Tufi S, Leonards P, Lamoree M, De Boer J, Legler J, Legradi J. Changes in neurotransmitter profiles during early zebrafish (Danio rerio) development and after pesticide exposure. Environ Sci Technol. 2016;50(6):3222–30.CrossRefGoogle Scholar
Højer-Pedersen J, Smedsgaard J, Nielsen J. The yeast metabolome addressed by electrospray ionization mass spectrometry: initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry. Metabolomics. 2008;4(4):393–405.CrossRefGoogle Scholar
Bajad SU, Lu W, Kimball EH, Yuan J, Peterson C, Rabinowitz JD. Separation and quantitation of water soluble cellular metabolites by hydrophilic interaction chromatography-tandem mass spectrometry. J Chromatogr A. 2006;1125(1):76–88.CrossRefGoogle Scholar
Gómez-Canela C, Miller TH, Bury NR, Tauler R, Barron LP. Targeted metabolomics of Gammarus pulex following controlled exposures to selected pharmaceuticals in water. Sci Total Environ. 2016;562:777–88.CrossRefGoogle Scholar
Gómez-Canela C, Prats E, Piña B, Tauler R. Assessment of chlorpyrifos toxic effects in zebrafish (Danio rerio) metabolism. Environ Pollut. 2017;220:1231–43.CrossRefGoogle Scholar
Lu H, Liang Y, Dunn WB, Shen H, Kell DB. Comparative evaluation of software for deconvolution of metabolomics data based on GC-TOF-MS. TrAC Trends Anal Chem. 2008;27(3):215–27.CrossRefGoogle Scholar
Asiago VM, Alvarado LZ, Shanaiah N, Gowda GAN, Owusu-Sarfo K, Ballas RA, et al. Early detection of recurrent breast cancer using metabolite profiling. Cancer Res. 2010;70(21):8309–18.CrossRefGoogle Scholar
Santos-Fandila A, Vázquez E, Barranco A, Zafra-Gómez A, Navalón A, Rueda R, et al. Analysis of 17 neurotransmitters, metabolites, and precursors in zebrafish through the life cycle using ultrahigh performance liquid chromatography-tandem mass spectrometry. J Chromatogr B: Anal Technol Biomed Life Sci. 2015;1001:191–201.CrossRefGoogle Scholar
Aragon A, Legradi J, Ballesteros-Gómez A, Legler J, van Velzen M, de Boer J, et al. Determination of monoamine neurotransmitters in zebrafish (Danio rerio) by gas chromatography coupled to mass spectrometry with a two-step derivatization. Anal Bioanal Chem. 2017;409(11):2931–9.CrossRefGoogle Scholar
Nusslein-Volhard C, Dahm R. Zebrafish: a practical approach. New York: Oxford University Press; 2002. p. 303.Google Scholar
Hildebrand DGC, Cicconet M, Torres RM, Choi W, Quan TM, Moon J, et al. Whole-brain serial-section electron microscopy in larval zebrafish. Nature. 2017;545(7654):345–9.CrossRefGoogle Scholar
Faria M, Garcia-Reyero N, Padrós F, Babin PJ, Sebastián D, Cachot J, et al. Zebrafish Models for human acute organophosphorus poisoning. Sci Rep. 2015;5 https://doi.org/10.1038/srep15591.
Core Team R. A language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2015.Google Scholar
Official Journal of the European Communities O (2002) Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Directive 2002/657/CE.Google Scholar
Alpert AJ. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. J Chromatogr. 1990;499:177–96.CrossRefGoogle Scholar
Tufi S, Lamoree M, de Boer J, Leonards P. Simultaneous analysis of multiple neurotransmitters by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A. 2015;1395:79–87.CrossRefGoogle Scholar
Stahnke H, Reemtsma T, Alder L. Compensation of matrix effects by postcolumn infusion of a monitor substance in multiresidue analysis with LC−MS/MS. Anal Chem. 2009;81(6):2185–92.CrossRefGoogle Scholar
Rezaei M, Sadeghian A, Roohi N, Shojaei A, Mirnajafi-Zadeh J. Epilepsy and dopaminergic system. Physiol Pharmacol. 2017;21(1):1–14.CrossRefGoogle Scholar
Lallement G, Denoyer M, Collet A, Pernot-Marino I, Baubichon D, Monmaur P, et al. Changes in hippocampal acetylcholine and glutamate extracellular levels during soman-induced seizures: influence of septal cholinoceptive cells. Neurosci Lett. 1992;139(1):104–7.CrossRefGoogle Scholar