Modulation of Cholinergic Signaling in Embryonic and Larval Zebrafish: Impacts on Circadian Rhythms, Learning, and Memory

Abstract

Approximately 10-20% of pregnant mothers continue to smoke during their pregnancy. Consequently, the unborn child, if it survives could suffer from low birth weight, infantile colic, impaired cognition, and sleeping problems. Paralleling those sleep problems resulting from developmental exposure to tobacco products, the prevalence of circadian rhythm (CR) disorders in adults are increasing. Specifically, cigarette smokers often report irregular rhythms and/or problems related to abnormal sleep. Nicotine is the main psychoactive ingredient in tobacco and is an agonist of nicotinic acetylcholine receptors (nAChRs). These receptors are found throughout the Central Nervous System (CNS) and are conserved from zebrafish to humans. Zebrafish were used to investigate the long term behavioral consequences of developmental nicotine exposure. Zebrafish (larval and adults) display robust and reliable behaviors such as escape swimming, learning, memory, and attention skills. These behaviors are all governed by the fish’s circadian clock. We first modulated cholinergic signaling during embryogenesis and determined the impact of that perturbation on CRs in larval and juvenile zebrafish, and learning and memory was analyzed in adult zebrafish. The CR was assessed by monitoring changes in locomotor activity in zebrafish entrained on a 24 hour light/dark cycle. An embryonic nicotine exposure altered the CR assessed in larval zebrafish, specific to nicotine concentration and the timing of the exposure. Adult zebrafish developmentally exposed to nicotine in exposure windows differing from those that impacted the CR, exhibited reduced recall ability and increased locomotor activity in separate exposure windows. In an acute, non-developmental nicotine exposure paradigm, the CR was dramatically altered by nicotine exposure in 5-6 days post fertilization (dpf) zebrafish. Our pharmacological experiments, also investigated potential nAChRs in mediating nicotine’s ability to alter the CR. The findings reported herein support epidemiological studies showing that children exposed prenatally to tobacco exhibit decreased cognitive skills, are hyperactive and have altered CRs. Our results showing that acute nicotine exposure can impact the CR of zebrafish are reminiscent of the irregular sleep cycles reported for tobacco smokers. Thus, more attention needs to be devoted to the public health implications of CR disorders and their relationship to the stimulant nicotine.