Embyronic Nicotine Exposure and Its Impact on Spinal Neuron Development in Zebrafish

Abstract

Nicotine, a drug of abuse, has been shown to have deleterious outcomes on nervous system development in vertebrates. Using zebrafish behavior as a diagnostic tool, we aimed to understand the effects of embryonic nicotine exposure on spinal neuron development. We initially quantified nicotine incorporation into zebrafish embryos. We found that only a fraction of the waterborne concentration of nicotine was incorporated into embryos. Also, when embryos were exposed to epibatidine and ABT 418, potent alpha 4/beta 2 nAChRs agonists, the embryos exhibited a robust swim-like behavioral response similar to that of nicotine. We then performed immunohistochemistry utilizing the mAB290 antibody and a zebrafish specific beta 2 antibody and found that Rohon-Beard (RB) neurons express the beta 2 subunit of nAChRs. Thus, we hypothesized that nicotine would have an effect on RB development. To test this, we first characterized RB neurons in embryonic zebrafish using known RB cellular markers. Early on, RB neurons form two rows in dorsal spinal cord then migrate medially towards the midline to form a linear row. We also examined the distribution of acetylated tubulin (aat), an indicator of programmed cell death (PCD) (Svoboda et al, 2001) in RB peripheral processes. We then evaluated the consequences of chronic nicotine exposure on RB neuron development. Altered distribution of aat in RB peripheral processes is a reliable marker for RB PCD. In nicotine exposed embryos, aat distribution appeared more continuous when compared to stage-matched controls. Also, RB migration to the midline was delayed. We conclude that chronic embryonic nicotine exposure can modulate RB PCD via nicotine binding to nAChRs expressed on RB neurons, thus play a neuroprotective role on RB development. Lastly, we discovered a novel zebrafish specific primary motoneuron marker. Upon using the beta 2 nAChR antibody, we noticed that not only RB neurons were labeled, but also primary motoneurons. To determine the stability and potential uses of this beta 2 nAChR antibody, we characterized beta 2 labeling in zebrafish ranging in age from 30 hpf to 12 dpf. We conclude that the beta 2 antibody specifically labels primary motoneurons consistently during development.