The proposed research will investigate the hypothesis that an important mechanism leading to certain of the CNS anomalies seen in the fetal alcohol syndrome (FAS) is a disruption of normal trophic interactions during neurogenesis. We propose that chronic prenatal ethanol treatment (CPET) results in a decrease in the synthesis, availability, delivery, or biological activity of normally occurring neurotrophic substances, such as nerve growth factor (NGF), or may alter the capacity of target neurons to respond appropriately to these factors. These developing relationships will be studied in the fetal and neonatal rat septo-hippocampal system. This system was chosen because its importance to normal cognitive functioning makes it likely to be involved in the severe intellectual impairment seen in FAS. Prior study has shown that the septal nuclei and their target hippocampus are selectively vulnerable to ethanol insult following adult chronic ethanol treatment (CET) in both humans and animal models. The hippocampus is similarly vulnerable to ethanol during development, and there is some limited evidence indicating that the basal forebrain nuclei are also damaged following CPET. The hippocampus synthesizes NGF, basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) and certain uncharacterized substances which provide normal trophic support for basal forebrain neurons. Initial experiments will examine developing basal forebrain neurons (of medial septal/vertical diagonal band of Broca nuclei [MS/VDB]) in control and CPET animals. Total population numbers will be compared, as will the number of cholinergic, GABAergic, and NGF-- receptor-positive neurons. In addition, autoradiographic studies will compare neuronal generation in MS/VDB in control and CPET groups. We will also examine the influence of CPET on the development of trophic activity in the hippocampal formation. In our prior studies in adult animals, we found that CET reduces trophic activity produced in the hippocampus. We will measure trophic activity of hippocampal extract from control and ethanol-exposed animals using both bioassays and ELISA quantifications (assessing NGF and bFGF content). We will also examine the influence of CPET on the capacity of septal and hippocampal neurons to respond normally to trophic factors (e.g., NGF, bFGF, hippocampal extract), and we will assess direct ethanol effects on cultured septal and hippocampal neurons, and septalhippocampal co-cultured explants, and the capacity of neurotrophic factors to ameliorate these effects. We will also investigate the ontogeny of cholinergic expression within the basal forebrain populations, in control and CPET animals. ChAT activity, high affinity choline uptake and ACh synthesis will be examined in vivo and in vitro. These studies have the potential to elucidate important cellular mechanisms underlying the devastating CNS damage seen in FAS, and could open the eventual possibility of therapeutic intervention in FAS mental retardation via neurotrophic factor replacement procedures.
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