Identifier

etd-06292010-170315

Degree

Doctor of Philosophy (PhD)

Department

Biological Sciences

Document Type

Dissertation

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 order to examine the role of the α2 nAChR subunit during embryonic development, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2 nAChR subunit altered the embryonic nicotine-induced swim-like behavior without affecting neuronal and muscle elements associated with this motor output. Since Rohon-Beard neurons and other spinal neurons expressed the α2 subunit during embryonic development, we propose that these spinal neurons express α2-containing nAChRs and are most likely involved in initiating and maintaining nicotine-induced motor output.

Date

2010

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Svoboda, Kurt

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