Ontogeny of a synaptophysin-mediated GABA transmission mechanism from the ciliary band-associated strand to the ciliary band during the development of the sea urchin Hemicentrotus pulcherrimus

Swimming activity of the sea urchin larva depends on ciliary beating primarily in the circumoral ciliary band (CB) and is regulated by several neurotransmitters, such as 5HT, dopamine and -aminobutyric acid (GABA). Accordingly, the larval swimming activity is severely inhibited by 3-mercaptopropionic acid [a glutamate decarboxylase (GAD) inhibitor]. Although GABA is detected in the CB, GAD is absent. GAD is expressed in the spatially segregated nearby ciliary band-associated strand (CBAS). Thus, it is assumed that GABA transmission extends from the CBAS to the CB. Here, we examined the synaptic transmission mechanism by focusing on the spatiotemporal expression pattern of synaptophysin (Syp), a synaptic vesicle glycoprotein. The sea urchin has a single copy of the Syp gene, which encodes a 266-amino acid protein with possibly 4 transmembrane domains. We generated an anti-Syp antibody (Ab). Immunoblotting (IB) detected Ab binding to a single band at approximately 38 kDa. Whole-mount immunohistochemistry (WMIHC) detected high intensity Ab binding to the CBAS. Syp was initially detected at the mesenchyme blastula stage (mBL) as a single band by IB. Accordingly, WMIHC detected Syp in the cytoplasm of small patches of several ectodermal cells at the mBL stage. Syp has also been detected in the cytoplasm of blastocoelar cells from the prism stage to the 2-arm pluteus stage (2aPL). By the 4aPL stage, Syp was expressed in the CBAS and was moderately expressed among the blastocoelar cells. Distinctive co-localization of GABA and Syp was not detected until the 2aPL stage. Beginning at the 4aPL stage, GABA was detected in the CB and Syp-positive puncta in the CBAS. In the CB, GABA was co-localized with the GABA-A receptor (GABAAR). Thus, the GABA signal may be transmitted from GAD in the CBAS through a Syp-mediated system to the CB and then, in the CB, to the basal body of the cilia through GABAAR.