Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors

We propose to use the zebrafish ( Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR β3, α2 and α7 subunits expressed in other species. RT-PCR was used to show that the β3 and α2 subunit RNAs were present in zebrafish embryos only 2–5 hours post-fertilization (hpf) while α7 subunit RNA was not detected until 8 hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish β3, α2, and α7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [ 3 H] -epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC 50 values of 28.6 pM and 29.7 nM and in 5 dpf embryos with IC 50 values of 28.4 pM and 8.9 nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.