Modulation of Cholinergic Signaling in Embryonic Zebrafish: Anatomical and Behavioral Consequences

Abstract

Smoking during pregnancy remains a major health concern in the United States. Nicotine is one of the main ingredients of cigarettes and a neurotoxicant which has been purported to affect many aspects of nervous system development causing long-term abnormalities in the offspring. We took advantage of the zebrafish model to investigate the effects of nicotine in nervous system anatomy and function. It was previously reported that embryonic nicotine exposure causes pathfinding errors in secondary motoneuron (SMN) axons through the activation of nicotine acetylcholine receptors (nAChRs). However, the nicotine-induced effects on SMN pathfinding coincided with muscle degeneration. We hypothesized that nicotine exposure could directly affect spinal neurons bypassing muscle degeneration effects. Focusing on the neural specific effects of nicotine exposure, we identified four unique, abnormal axonal pathfinding errors which were caused by the exposure. We then demonstrated that the nicotine-induced motoneuron and muscle effects uncoupled in a dose-dependent manner. Showing that embryonic nicotine exposure could directly alter SMN axon pathfinding, we hypothesized that these nicotine-induced effects would persist into later stages of life. To test this hypothesis, we developed a live imaging method where transgenic zebrafish expressing green fluorescent protein in SMNs and their axons could be visualized over the course of several weeks. We demonstrated that embryonic nicotine exposure caused axonal pathfinding errors that persisted into adulthood. We then hypothesized that these anatomical changes would potentially coincide with physiological changes that would ultimately result in altered behavior of the adult. To test this hypothesis, we developed a method in which the swimming behavior in freely swimming adult zebrafish was examined using electromyographic recordings from the axial muscles of adult zebrafish exposed to nicotine as embryos. Our analyses revealed that several distinct features indicative of swimming behavior in zebrafish were altered by transient nicotine exposure during embryogenesis. Changes in the overall swimming frequency, the relationship between burst duration and cycle time, and the alternating pattern of activity were all altered in adult fish. These may be related to alterations in the physiological properties of interneurons within the spinal circuitry that govern locomotor output. The actions of nicotine are thought to be mediated through the activation of nAChRs. We investigated the expression pattern of the α2 nAChR subunit as evidence form others suggested it would be expressed by motoneurons and/or interneurons. Using a zebrafish-specific antibody, we showed that the α2 nAChR subunit exhibited a diverse and developmentally regulated pattern of expression. In