Our past studies as well as those of others have indicated that alcohol abuse leads to a loss of docosahexaenoate (DHA), the major polyunsaturate in the nervous system. Nutritional inadequacies, particularly during early development, may also lead to such losses in this essential fatty acid. In following up this work, it is important to establish what losses in physiological functions are caused by the loss of DHA in various organ systems. In a collaboration with several investigators at Wayne State University, the relationships of alcohol intake during pregnancy is related to the mother's and newborn infant's essential fatty acid and vitamin status. Dietary information is collected from the mothers in order to ascertain whether alcohol affects food selection or has a more direct metabolic effect in mediating potential losses in blood stream essential fats. Initial work on 225 women at their first prenatal care visit indicates that alcohol intake inversely correlates with plasma vitamin E content. Complex interactions appear to be taking place between essential fatty acid levels in the mothers plasma and her history of alcohol consumption during pregnancy. Samples obtained at the time of birth including mothers blood components and cord blood and artery/vein samples are currently being analyzed for essential fatty acids. In addition, plasma levels of folate metabolites have been measured in the pregnant women at the 24 week stage of gestation. A novel application to the field of essential fatty acid biology was made with the introduction of olfactory-based learning and memory-related tasks for brain function assessment. This modality was used since Slotnick has reported that rats are capable of high level learning of olfactory based tasks of a nature usually only ascribed to non-human primates or higher mammals. Our principal findings are that there is a poorer performance in the acquisition of olfactory set learning in rats where brain and olfactory bulb DHA was lowered thru dietary insufficiency. That is, after the rats had acquired the task, they were over-trained in order to determine whether they could achieve the learning set, i.e., make zero or only one mistake in the first twenty trials after an information trial in a two-odor discrimination task. Rats given a safflower oil based diet for two generations were significantly poorer in this regard than rats to which oils containing alpha-linolenate and DHA were added. Animals with lower levels of brain DHA performed more poorly on spatial maze tasks using the Morris Water Maze. The n-3 deficient rats swam longer and at a higher rate, but found the platform with a longer latency. In a memory retention trial, n-3 deficient rats performed significantly worse than the n-3 adequate group, especially when deprived for three generations. Although n-3 deficient rats perform more poorly, it cannot be ascribed to lower activity or motivation as general motor activity was not different between groups and there was no difference in a progressive-ratio licking task in which animals worked for a water reward. Also, the n-3 deficient rats sampled the odors longer than the DHA-adequate animals but still made more subsequent total errors. The n-3 deficient rats were examined for changes in brain morphology using quantitative stereological techniques. Initially, studies focused on hippocampal morphology. After fixing and cresyl violet staining, a variety of features were quantified in rats fed the n-3 deficient or adequate diets for three generations. No statistically significant differences were observed in volume, density or total number of perikaryal neurons in the hippocampus. However, perikaryal size in the septal area of the CA1 and CA2/3 fields of the hippocampus were greater in the n-3 adequate (containing DHA) group relative to the deficient group. Similar findings have now replicated and extended this finding as similar changes in cell body size have now been observed in the hippocampus, hypothalamus, subfornical organ, and in the parietal and piriform cortex.. The potential implications for infant nutrition are great as it may be surmised that infants fed formulas available in North America may have similar structural changes in their brains. Once these deficiencies in brain function were established relating to DHA status, it was of interest to determine whether they were reversible. For this purpose, n-3 deficiency was induced with a safflower oil-based diet over three generations. The offspring were then repleted with a diet containing flax oil (supplying alpha-linolenate) and DHA at various times in development, i.e., at birth, weaning and young adulthood (7 wks) and tested on spatial task performance beginning at 9 or 13 weeks of age. Our findings were that rats repleted at birth (by cross-fostering) or at weaning had substantially recovered their brain DHA by 7 weeks of age and their spatial task performance was similar to that of n-3 adequate animals. When animals were repleted at young adulthood, their degree of recovery in both respects depended upon the duration of dietary repletion. When assessed at 9 weeks of age, neither brain DHA nor spatial task performance had recovered. However, when assessed at 13 weeks, DHA had substantially recovered and spatial task performance had also substantially recovered such that it was not significantly different from that of n-3 adequate animals. These are important results as they indicate that intervention with a DHA-rich diet can reverse at least some of the adverse consequences for brain development caused by an inadequate diet early in development. In order to expedite future studies of n-3 deficiency and to make them more relevant to the human condition, a model of deficiency based on the first generation was developed. This employs the auto-rearing method of Hoshiba for rat pups starting from the first day of life. This approach has now succeeded in producing live animals and ones extremely deficient in brain DHA when an artificial milk is fed that has very low levels of n-3 fats. Also, animals have been weaned onto the same pelleted diets with respect to the n-3 fatty acid content and raised until early adulthood, 8 weeks of age. This has made possible behavioral testing of both spatial task performance as well as an olfactory cued operant task. In the current reporting period, these studies were extended to demonstrate that young rats only 6 weeks of age, were capable of performing olfactory discriminations and reversal learning. It was necessary to limit the water, used for reward in this operant task, only with respect to the length of time access was allowed rather than to induce a water deficit during this period of rapid growth. Preliminary data have been produced on the elevated plus maze indicating greater anxiety of rats deficient in brain DHA. Also, the Morris water maze data indicates poorer performance in DHA-deficient animals and poorer spatial memory. Rats of 29 days in age that were fed the n-3 deficient diet were found to exhibit a 73% loss in brain DHA. Thus infants who receive no n-3 fats from infant formula may be expected to also have a severe drop in their brain DHA during this period of rapid brain growth. This model will also provide for a great savings in the use of animals and in the time needed to produce a model of n-3 deficiency. This apparatus will make possible a new model of fetal alcohol syndrome where animals can be given alcohol from the first days of life.
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