Novel Transgenic Zebrafish Lines to Study the CHRNA3-B4-A5 Gene Cluster

Acetylcholine (ACh), a vital neurotransmitter for both the peripheral (PNS) and central nervous systems (CNS), signals through nicotinic ACh receptors (nAChRs) and muscarinic ACh receptors (mAChR). Here, we explore the expression patterns of three nAChR subunits, chrna3, chrnb4, and chrna5, which are located in an evolutionary conserved cluster. This close genomic positioning, in a range of vertebrates, may indicate co-functionality and/or co-expression. Through novel transgenic zebrafish lines, we observe widespread expression within both the PNS and CNS. In the PNS, we observed expression of chrna3^tdTomato, chrnb4^eGFP, and chrna5^tdTomato in the intestinal enteric nervous system; chrna5^tdTomato and chrnb4^eGFP in sensory ganglia of the lateral line; and chrnb4^eGFP in the ear. In the CNS, the expression of chrnb4^eGFP and chrna5^tdTomato was found in the retina, all three expressed in diverse regions of the brain, where a portion of chrna3^tdTomato and chrnb4^eGFP cells were found to be inhibitory efferent neurons projecting to the lateral line. Within the spinal cord, we identify distinct populations of chrna3^tdTomato-, chrnb4^eGFP-, and chrna5^tdTomato-expressing neurons within the locomotor network, including dmrt3a-expressing interneurons and mnx1-expressing motor neurons. Notably, three to four primary motor neurons per hemisegment were labeled by both chrna3^tdTomato and chrnb4^eGFP. Interestingly, we identified an sl-type secondary motor neuron per hemisegement that strongly expressed chrna5^tdTomato and co-expressed chrnb4^eGFP. These transgenic lines provide insights into the potential roles of nAChRs within the locomotor network and open avenues for exploring their role in nicotine exposure and addiction in a range of tissues throughout the nervous system.