EXPERIMENTAL FETAL ALCOHOL SYNDROME

对胎儿酒精综合征（FAS）患者和实验性FAS大鼠的研究表明，早期接触乙醇会严重影响大脑结构和功能。乙醇引起的缺陷包括小脑畸形、大脑皮层变薄和皮层神经元数量减少。这些发现可能源于一个单一的原因——乙醇引起神经元死亡的毒性作用。我们将验证以下假设：乙醇诱导的神经元死亡是由于干扰了神经生长因子（NGF）的促生存活性，这种干扰导致编码已知和/或新蛋白质的基因表达改变。体内研究将检验产前暴露于乙醇对NGF表达和死亡相关蛋白（特别是p75和bcl蛋白）表达的影响。p75作为NGF的低亲和力受体，受乙醇处理的影响，bcl蛋白可以抑制（如bcl-2）或促进（如bax）神经元死亡。这些研究将集中在三叉/体感觉系统的三个组成部分：体感觉皮层、腹基底丘脑和三叉神经的主要感觉核。一系列的体外研究将验证上述假设以及NGF和乙醇治疗相互拮抗的推论。两种类型的细胞将被检查：纯化的皮层神经元和有条件永生化的神经母细胞。细胞将单独在无血清培养基中培养，在添加了NGF或乙醇的培养基中培养，或在添加了NGF和乙醇的培养基中培养。该设计将用于确定NGF和乙醇(a)对神经元存活（细胞计数、胸苷镍端标记程序（TUNEL）和电子显微镜）和(b)对bcl基因产物表达的影响。随后，NGF和乙醇对基因表达的影响将使用依赖于诱导mrna差异显示的技术来确定。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，其中死亡细胞被积极识别（例如，用TUNEL）。新开发的探针在三叉/体感觉系统中的时空表达将在体内进行检测。动物将在出生前暴露于乙醇中，它们的后代将被检测不同mrna的表达。本实验探讨了FAS的机制，并验证了与FAS相关的中枢神经系统缺陷是由ngf介导的蛋白表达改变引起的假设。

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.