EXPERIMENTAL FETAL ALCOHOL SYNDROME

对患有胎儿酒精综合征的人和实验性胎儿酒精综合征大鼠的研究表明，早期接触乙醇会深刻影响大脑结构和功能。酒精引起的缺陷包括小脑，大脑皮质变薄，皮质神经元数量减少。这些发现可能是由单一原因造成的--乙醇导致神经元死亡的毒性效应。我们将验证这样的假设，即乙醇诱导的神经元死亡是由于干扰神经生长因子(NGF)的存活促进活性导致的，并且这种干扰导致编码已知和/或新蛋白质的基因的表达改变。活体研究将检测产前接触乙醇对NGF表达和死亡相关蛋白表达的影响，特别是p75和bc1蛋白。P75作为神经生长因子的低亲和力受体，受乙醇处理的影响，bc1蛋白可抑制(如bcl2)或促进(如bax)神经元死亡。这些研究将集中在三叉神经/躯体感觉系统的三个组成部分：躯体感觉皮质、丘脑腹侧基底核和三叉神经的主要感觉核。一系列体外研究将检验上述假设和NGF和乙醇处理相互拮抗的推论。将检查两种类型的细胞：纯化的皮质神经元培养和条件永生化的神经母细胞。细胞将在单独的无血清培养液中培养，或在添加NGF或乙醇的培养液中培养，或在含有NGF和乙醇的培养液中培养。本设计将用于确定NGF和乙醇(A)对神经元存活(细胞计数、胸腺嘧啶核苷缺口末端标记程序(TUNEL)和电子显微镜)和(B)对bc1基因产物表达的影响。随后，将使用一种依赖于诱导的mRNAs差异显示的技术来确定NGF和乙醇对基因表达的影响。这些新蛋白在活的和死亡的神经元中的表达将使用双标记技术来确定，其中死亡的细胞得到了肯定的鉴定(例如，用TUNEL)。新开发的探针在三叉神经/体感系统中的时空表达将在体内进行检测。动物将在产前暴露在乙醇中，它们的后代将被检测差异显示的mRNAs的表达。建议的实验探索了Fas的机制，并验证了与Fas相关的中枢神经系统缺陷是由于NGF介导的蛋白表达改变所致的假说。

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.