Cloning and spatiotemporal expression of zebrafish neuronal nicotinic acetylcholine receptor alpha 6 and alpha 4 subunit RNAs

Abstract

Acetylcholine plays an important role in regulation of nervous system development and function. We are developing zebrafish (Danio rerio) as a model system to study the role of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes in development and the effects of nicotine on the developing vertebrate nervous system. We previously characterized the expression of several zebrafish nAChR subunits. To further develop the zebrafish model, here we report a study on the molecular characterization of two additional nAChR subunit genes, designated chrna6 and chrna4. Both zebrafish nAChRs have a high degree of sequence identity to nAChRs expressed in a variety of mammalian species. Reverse transcription polymerase chain reaction was used to show that both nAChR subunit RNAs were expressed early in zebrafish development, with the chrna4 transcript present at 3 hours postfertilization (hpf) and the chrna6 RNA present at 10 hpf. In situ hybridization was used to localize chrna6 and chrna4 RNA expression in 24, 48, 72, and 96 hpf zebrafish. The chrna6 and chrna4 RNAs were each expressed in a unique pattern, which changed during development. At various ages, chrna6 was expressed in Rohon-Beard sensory neurons, trigeminal ganglion, retina, and the pineal gland. Most notably, chrna6 was expressed in catecholaminergic neurons in the midbrain, but was also present in noncatecholaminergic cells in both midbrain and hindbrain. The expression of chrna6 RNA in catecholaminergic cells supports the use of zebrafish as a valid model system to better understand the molecular basis of cholinergic regulation of dopaminergic signaling and the role of alpha6-containing nAChRs in Parkinson's disease. The most notable chrna4 expression was in neural crest cells at 24 hpf and reticulospinal neurons in hindbrain at 48 hpf. chrna4 RNA exhibited a widespread and robust expression pattern in the midbrain in 72 hpf and 96 hpf zebrafish.