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 beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.
Zirger
Jeffrey M
JM
Department of Neuroscience, College of Medicine and Public Health, The Ohio State University, 4068 Graves Hall, 333 West Tenth Avenue, Columbus, OH 43210, USA.
Beattie
Christine E
CE
McKay
Dennis B
DB
Boyd
R Thomas
RT
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000378:metabolism; D015870:Gene Expression; D009474:Neurons; D018722:Nicotinic Agonists / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D064569:alpha7 Nicotinic Acetylcholine Receptor
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D021122:Protein Subunits; D011725:Pyridines; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D029961:Zebrafish Proteins; D064569:alpha7 Nicotinic Acetylcholine Receptor; C488354:chrna2a protein, zebrafish; C488355:chrna7 protein, zebrafish; C488356:chrnb3a protein, zebrafish; C082748:epibatidine