The proposed training is designed to provide the candidate with a background in animal cognition and testing as well as training in molecular mechanisms of development. The candidate is a well-trained developmental cognitive neuroscientist with a dedication to at-risk children. However, human research is limiting in that mechanisms cannot be established due to the lack of available tissue. The training proposed in this application will facilitate the candidate's pursuit of her long-term goal of being able to ask scientific questions by moving from the clinic to the bench and back again. Specifically, the candidate will work on the mechanisms involved in the effects of fetal alcohol exposure. This preventable, highly prevalent developmental phenomenon is characterized by a constellation of adverse outcomes including neurodevelopmental abnormalities, growth retardation, and facial structure anomalies. Scientists working with animal models have provided evidence that choline supplementation given concomitant with or after fetal alcohol exposure mediates the effects. The proposed research will establish the mechanisms involved in this mediation by describing any epigenetic modifications and will extend previous research by testing choline as a preventative measure, giving it before and during alcohol exposure in a rat model. In Experiment 1, the candidate will develop a novel choline-supplemented liquid diet for the rats. This diet will be important in the avoidance of the stress of injections that is inherent in cholin administration. The goal of Experiment 1 is to determine if the taste of choline will deter the rat from consuming as much ethanol as the ethanol-only group. If so, subcutaneous injections of choline and vehicle will be necessary. In Experiment 2, pregnant rats will be divided into 3 experimental groups: ethanol-exposed, choline-supplemented, and ethanol-exposed with choline supplementation. Each experimental group will have a matching control group, and an unhandled pellet fed control will be included for a total of 7 diet groups. One male pup from each litter (n=10) will comprise groups for analyses. Hippocampal slices, blood DNA, and brain DNA will be collected on GD 18 (after cell proliferation in hippocampus), PD 45 (prior to Morris water maze training), and PD 54 (after Morris water maze training). Hippocampal slices and blood will be assessed with bisulfite pyrosequencing (epigenetics) and real-time RT-PCR (gene expression) for a selection of genes related to hippocampal development, choline, and alcohol metabolism. Protein levels will be assessed by immunolabeling of frozen brain sections. The research will be completed with a mentoring team comprised of top experts in the field: Steven Zeisel (choline and epigenetics), Jennifer Thomas (animal models of fetal alcohol exposure and mediation of same with choline), and Phillip May (sequelae of fetal alcohol effects in humans). The candidate and two of the mentors are on the faculty of the University of North Carolina at Chapel Hill Nutrition Research Institute (NRI) on the new North Carolina Research Campus in Kannapolis, North Carolina. Having only opened in 2008, this campus is outfitted with state-of-the-art facilities. Laboratories in the NRI are equipped with the newest of DNA sequencing, genotyping, and gene expression equipment. The David H Murdock Research Institute (DHMRI) provides over 110,000 square feet of instrumentation, resident expertise, and well-equipped "-omics" laboratories. In addition, the DHMRI maintains a 40,000 square foot vivarium with top-of-the-line equipment. All equipment needed for this project is available on this campus;many scientists are housed here and are available to provide expert support to the candidate. The results of these studies will inform an R01 application (candidate's short-term goal) in which the candidate will propose an intervention to determine the effects of choline supplementation in human fetuses who are at risk for fetal alcohol exposure. This work is integral to the understanding of the fetal alcohol exposure phenotype and will be the initial steps toward the use of a nutrient - choline - to mediate or even prevent the effects of fetal alcohol. The candidat has demonstrated her commitment to at-risk children. The proposed training will provide her with a deeper understanding of the mechanisms involved in developmental phenomenon thereby strengthening her ability to form and test hypotheses. As a result of being able to move between the clinic and the bench, the candidate will build a strong, independent research program.
The project will explicate the mechanisms involved in the mediation of fetal alcohol effects by choline supplementation in rats. Epigenetic modifications and hippocampal dysfunction will be explored. The results will be the basis for a clinical trial in which choline will be tested for its ability to prevent fetal alcohol effects in at-risk infants.