Development of the γ-amino butyric acid (GABA)-ergic signaling system and its role in larval swimming in sea urchin

Abstract

Summary The present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning, namely, glutamate decarboxylase (Hp-gad), the GABAA receptor (Hp-gabrA), and GABAA receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2-day-post-fertilization (dpf) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by reverse transcription-polymerase chain reaction during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2-dpf pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA-immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4-dpf plutei, but strongly from fertilized eggs to 2-dpf plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the 2-arm pluteus stage. Hp-gabarap transcription and protein expression were detected from the swimming blastula stage. GABAA receptor inhibition by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17-dpf and 34-dpf plutei) than in younger ones (1-dpf prism larvae).