Immunochemical localization of GABAA receptor subunits in the freshwater polyp Hydra Vulgaris (Cnidaria, Hydrozoa)

Gamma-aminobutyric acid (GABA) is the most widely distributed inhibitory neurotransmitter in both vertebrate and invertebrate species. GABA receptors have been described in several invertebrate species of different phyla. Despite the considerable variability of results, it is becoming clear that in many invertebrate species GABA receptors present a pharmacology comparable to that of mammalian ones without fitting precisely into the classification developed for mammalian brain. We have demonstrated that GABA is present in Hydra tissues and that Hydra responds to GABA positive allosteric modulators. To approach the molecular characterization of GABA receptors, we examined the occurrence and distribution of GABAA receptor subunits in Hydra polyps. The presence and localization of GABAA receptor subunits in Hydra was examined by Western Blot in membrane preparations and immunohistochemical analysis of whole mount preparations. The presence of GABAA receptor subunits in Hydra membrane preparations, was tested using different antibodies against a1, a2, a3, b1, b2, b3, g1, g2, g3, d, r2 and e subunits, each probed with the corresponding peptide. In membranes incubated with the a3 antibody the unique band inhibited by the specific peptide appeared at 35 kDa. Protein bands blotted with the b1 antibody occurred at 60 kDa, while those blotted with g3 and d antibodies revealed bands at approximately 50 kDa and 52 kDa, respectively. None of the other subunit antibodies examined revealed protein bands in Hydra membranes. Immunohistochemical analysis by a3 and b1 antibodies revealed a diffuse immunoreactivity in tentacles, hypostome, and upper part of the gastric region. In the peduncle, double labelling clearly showed co-localization of a3 and b1 immunoreactivity on a circular structure above the foot; in the tentacles, occasional cell bodies also exhibited double staining in the form of patches. By contrast both g3 and d antibodies obtained a strong labelling in the lower gastric region and in the peduncle; occasional immunoreactive cells were observed in the hypostome and tentacles. Double labelling showed co-localization of a3/g3 immunoreactivity in granules or cells in the gastric region, and in the peduncle, where circular fibers above the foot were clearly double-stained. Finally, co-localization of a3 and d immunoreactivity was observed in cells of the tentacles, the gastric region and the peduncle. These data indicate that populations of specific subunits of the GABAA receptor are present in Hydra vulgaris, the most primitive metazoans to have developed a nervous system.