Effect of Nicotine on the Zebrafish Brain: A Tale of Two Methods

The use of nicotine, a known anxiolytic with addictive properties, is prevalent; however, the effects on the brain are not fully elucidated. Previous studies investigating the effects of nicotine on the brain have been limited to developmental models. In this two-part study, we investigated the effect of nicotine on protein kinase B (AKT) and glycogen synthase kinase-3β (GSK-3β) in the brain of adult zebrafish. AKT plays a role in neuronal growth, plasticity, and metabolism. AKT is activated via phosphorylation on Ser473 or Thr308 by growth factors and increased neuronal activity. AKT regulates constitutively active GSK-3β via phosphorylation on Ser9. This is important in neurophysiology, because unregulated GSK-3β is associated with cell death, dysregulated metabolic function, and neuro-inflammation. Two protocols were used for measuring the effect of nicotine on the adult zebrafish brain: an in-vivo (immersion) treatment, and an ex-vivo treatment. The protocol for immersion experiment included three groups: home (naive) control, vehicle (EtOH) control, and 15uM nicotine treatment. Fish were treated with vehicle or nicotine via immersion for five minutes, followed by dissection of the telencephalon and optic tectum, brains of naive fish were harvested with no treatment. The protocol for the ex-vivo experiment included three groups: control, nicotine (0.1 g/mL) and nicotine plus mecamylamine, a nicotinic receptor inhibitor (0.2 g/mL). Brains were harvested and placed into resting chambers for 60 minutes with oxygenation, followed by treatment for 10 minutes. The telencephalon and optic tectum were separated, and tissues were prepared for western blot. Results showed no significant changes in pAKT after immersion treatment. Ex-vivo method results were significant for increased pSer473AKT in telencephalon and optic tectum, which was significantly attenuated by mecamylamine co-treatment. Our results indicate treatment method is important to consider when investigating the effect of nicotine on the brain