The long term objectives of this research are to identify and characterize the cellular mechanisms underlying ethanol induced changes in neuronal development, and to assess the role of these changes in the etiology of CNS abnormalities associated with Fetal Alcohol Syndrome (FAS). Key neuropathologic features of FAS include altered neuronal morphogenesis and synapse formation in the hippocampus. Recent studies have elucidated some of the molecules and processes responsible for the distinct growth characteristics of axons and dendrites, thus providing the basis for novel experiments to determine the mechanisms underlying ethanol's disruption of neuronal development. The use of primary cultures of embryonic rat hippocampal pyramidal neurons is integral to the objectives of the project. Neurons in these cultures develop axons, dendrites, and synapses in a sequence of events that mimics their development in vivo. Experiments in this proposal are designed to use immunofluorescent cytochemistry coupled with quantitative morphometric analysis, time lapse videomicroscopy, and Fura-2 intracellular free calcium measurements in the following specific aims: (1) Compare the sensitivity of cultured hippocampal neurons exposed to ethanol at different times relative to development of axons, dendrites and synapses, (2) Distinguish whether ethanol induced changes in neuronal development result from direct effects of ethanol on neurons, or indirect effects on neurons mediated by astrocytes, (3) Determine whether ethanol's effects on process outgrowth involve altered regulation of intracellular calcium levels. The results of these studies will establish whether a disruption of process outgrowth and molecular compartmentalization is a key aspect of ethanol's neurodevelopmental toxicity and will provide important insight into the mechanism(s) underlying these actions. This fundamental knowledge can be expected to provide a basis for improving the identification and treatment of affected individuals.
