FETAL ALCOHOL EXPOSURE, NMDA RECEPTORS AND NEUROSTEROIDS

产前酒精暴露会严重影响大脑，导致神经行为缺陷，对以后的生活产生破坏性影响。因此，重要的是要制定治疗措施，尽量减少胎儿酒精暴露的这些有害影响。了解乙醇产前效应的细胞和分子机制是开发此类治疗方法的先决条件。在子宫内乙醇暴露影响的神经元蛋白中，NMDA受体（NMDA-Rs）在神经元发育、可塑性和死亡中起重要作用。许多研究表明，产前乙醇暴露影响NMDA-R;然而，乙醇这种影响的机制尚未阐明。我们的假设是，产前乙醇暴露改变了神经类固醇的行动，这是内源性的NMDA-Rs的调节剂。研究设计包括大鼠在妊娠期间暴露于导致血液酒精浓度接近人类法律的中毒限值（约0.08 g/dl）的饮食。目的#1是测量胎儿乙醇暴露对不同类别神经类固醇调节NMDA-Rs的影响。我们将使用电生理学技术来研究已被证明可以增强或抑制NMDA-R功能的神经类固醇的作用。这些研究将在来自新生大鼠的培养海马神经元以及来自成年大鼠的海马切片中进行。目的#2是确定产前乙醇诱导的神经甾体对NMDA-R的调节作用改变的机制。亚基组成和蛋白磷酸化是影响NMDA-Rs对神经甾体敏感性的因素。我们将使用Western印迹，放射免疫组织化学和单细胞RT-PCR技术来评估亚基组成。我们将测试蛋白激酶和/或磷酸酶的抑制剂和/或激活剂对培养的神经元和海马切片中NMDA-Rs的神经类固醇敏感性的影响。目的#3是确定胎儿酒精暴露对调节NMDA-R功能的神经类固醇海马水平的影响。我们将使用放射免疫测定法测定胚胎、新生儿和成年大鼠中的大肠杆菌水平。重要的是，研究表明，胎儿酒精暴露产生的产前压力会导致对神经类固醇的神经行为影响的反应性降低。我们建议做的实验可以增加我们对乙醇诱导的神经类固醇敏感性降低机制的理解。此外，这些实验将增加我们对海马NMDA-R在酒精相关出生缺陷发病机制中所起作用的认识。

The brain is severely affected by prenatal alcohol exposure resulting in neurobehavioral deficits that have devastating effects later in life. Therefore, it is important to develop therapeutic measures that minimize these noxious effects of fetal alcohol exposure. Understanding the cellular and molecular mechanism of the prenatal effects of ethanol is a prerequisite to developing such treatments. Among the neuronal proteins affected by in utero ethanol exposure are the NMDA receptors (NMDA- Rs), which play important roles in neuronal development, plasticity and death. A number of studies indicate that prenatal ethanol exposure affects NMDA-Rs; however, the mechanism of this effect of ethanol has not yet been elucidated. Our hypothesis is that prenatal ethanol exposure alters the actions of neurosteroids, which are endogenous modulators of NMDA-Rs. The research design includes exposure of rats during pregnancy to a diet that results in blood alcohol concentrations near the legal intoxication limit in humans (approximately 0.08 g/dl). Aim #1 is to measure the effects of fetal ethanol exposure on the regulation of NMDA-Rs by different classes of neurosteroids. We will use electrophysiological techniques to study the actions of neurosteroids that have been shown to either potentiate or inhibit NMDA-R function. These studies will be performed in cultured hippocampal neurons from neonatal rats as well as hippocampal slices from adult rats. Aim #2 is to determine the mechanism of the prenatal ethanol-induced alterations o the modulatory actions of neurosteroids on NMDA-Rs. Subunit composition and protein phosphorylation are factors that could regulate the sensitivity of NMDA-Rs to neurosteroids. We will use Western blot, radioimmunohistochemistry and single-cell RT-PCR techniques to assess subunit composition. We will test the effect of inhibitors and/or activators o protein kinases and/or phosphatases on neurosteroid sensitivity of NMDA-Rs in cultured neurons and hippocampal slices. Aim #3 is to determine the effect of fetal alcohol exposure on the hippocampal levels of neurosteroids that regulate NMDA-R function. We will use radioimmunoassays to determine levels in hippocampi from embryos, and neonates as well as adult rats. Importantly, studies have shown that prenatal stress produced by fetal alcohol exposure results in a reduction in the responsiveness to the neurobehavioral effects of neurosteroids later in life. The experiments that we propose to do could increase our understanding of the mechanism of this ethanol-induced decrease in sensitivity to neurosteroids. Moreover, these experiments will increase our knowledge of the roles played by hippocampal NMDA-Rs in the pathogenesis of alcohol-related birth defects.