An HPLC analysis of neurotransmitters in sea urchin larvae

Abstract

Classical neurotransmitters such as acetylcholine (ACh), biogenic amines and ϒ-aminobutyric acid (GABA) have been shown to mediate various forms of intercellular signalling in marine invertebrates including sea urchin larvae (Buznikovet al., 1996). The presence of amines and GABA in the nervous system of sea urchin larvae has been demonstrated by a cytochemical method at the gastrula stage (Bisgrove & Burke, 1987). However, few quantitative analyses of neurotransmitters have been attempted in the embryonic sea urchin. To accomplish this during the period of embryogenesis, in the present study we attempted to make homogenate samples adequate for HPLC analysis and to determine the levels of neurotransmitter in their extracts.We established an HPLC protocol using a batch of four-armed sea urchin larvae, and detected neurotransmitters (per milligram wet weight) in the homogenate of pluteus larvae ofHemicentrotus pulcherrimus(1.5–3.5 pmol dopamine, 0.22–0.51 pmol serotonin, 4.4–40 pmol ACh, 0.28–0.36 nmol glutamate, 20–23 nmol glycine) (Figs. 1, 2). GABA was not detected (detection limit, 10 pmol/10 μl injection). We also observed glyoxylic-acid-induced yellowish-green monoamine fluorescence in the larval nervous system. Serotonin has been a leading candidate neurotransmitter in sea urchin larvae (Nakajimaet al., 1993).