Cloning, expression and functional analysis of the zebrafish neuronal nictotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand-gated ion channel superfamily of receptors. Each nAChR is composed of five subunits that assemble in combinations around a central channel to give the nAChRs many diverse properties. These receptors are classified into two groups, based on their location at the neuromuscular junction (NMJ) or in the nervous system.; The primary function of neuronal nAChRs is to modulate synaptic transmission. Recent findings indicate that neuronal nAChRs play a role in the development of the nervous system. Because the nAChR has been implicated in nervous system development, the zebrafish (Danio rerio) has shown incredible potential as a model for the examination of the nAChR in these events.; This manuscript is dedicated to describing the initial studies of the neuronal nAChR in the zebrafish model. Using RT-PCR and 5′ and 3′ RACE, our laboratory has cloned and sequenced the first neuronal nAChR subunit genes. To date, these include the α2, β3, α7 and α8 nAChR subunits. Cloning of potential zebrafish nAChR α3 and β4 subunit cDNAs is also currently underway. Using RT-PCR, the time course of expression of the zebrafish nAChR α2, β3 and α7 RNAs has been determined. Each of these subunits was also localized using in situ hybridization with zebrafish embryos at specific time points. Ligand binding studies using the nAChR agonist epibatidine demonstrated the presence of assembled receptor subtypes by 2 and 5 days post fertilization (dpf), and expression of the zebrafish nAChR α7 subunit RNA in Xenopus oocytes provided evidence that these receptors are functional. Finally, apoptosis assays have been performed using nicotine and other cholinergic agents during different developmental time points to provide preliminary evidence of the effect nicotine may have on nervous system development.; The work performed by our laboratory has provided the initial studies of the zebrafish neuronal nAChR, and provides further evidence for the use of this model in the study of the nAChR in development.