Studies of humans with fetal alcohol syndrome (FAS) and rats with experimental FAS show that brain structure and function are profoundly affected by early exposure to ethanol. Ethanol-induced defects include microencephaly, a thinner cerebral cortex, and reductions in the number of cortical neurons. These findings may result from a single cause- the toxic effects of ethanol to cause neuronal death. We will test the hypotheses that ethanol-induced neuronal death results from interference with the survival-promoting activities of nerve growth factor (NGF) and that this interference results in the altered expression of genes coding for known and/or novel proteins. In vivo studies will examine the effect of prenatal exposure to ethanol on the expression of NGF and on the expression of death- associated proteins, specifically p75 and bcl proteins. p75 serves as the low affinity receptor for NGF and is affected by ethanol treatment, and bcl proteins can repress (e.g., bcl-2) or facilitate (e.g., bax) neuronal death. These studies will focus on three components of the trigeminal/somatosensory system: the somatosensory cortex, the ventrobasal thalamus, and the principal sensory nucleus of the trigeminal nerve. A series of in vitro studies will test the above hypotheses and the corollary that NGF and ethanol treatments are mutually antagonistic. Two types of cells will be examined: purified cultures of cortical neurons and conditionally immortalized neuroblasts. Cells will be raised in a serum-free medium alone, a medium supplemented with NGF or ethanol, or a medium with NGF and ethanol. This design will be used to determine the effects of NGF and ethanol (a) on neuronal survival (cell counts, thymidine nick-end labeling procedure (TUNEL) and electron microscopy) and (b) on the expression of bcl gene products. Subsequently, the effects of NGF and ethanol on the gene expression will be determined using a technique relying on the differential display of induced mRNAs. The expression of these novel proteins in living and dying neurons will be determined using double-labeling techniques in which the dying cells are positively identified (e.g., with TUNEL). The spatiotemporal expression of the newly developed probes in the trigeminal/somatosensory system will be examined in vivo. Animals will be exposed to ethanol prenatally and their offspring will be assayed for the expression of the differentially displayed mRNAs. The proposed experiments explore a mechanism of FAS and test the hypothesis that CNS defects associated with FAS result from alterations in NGF-mediated protein expression.
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