Molecular biological approach to explore the cause of brain damage after prenatal ethanol exposure

为了确定产前乙醇暴露后导致大脑变化的基因和因素，我们检测了乙醇暴露大鼠脑中的基因表达和神经递质或神经营养因子的测定。利用抗5-羟色胺抗体进行免疫组化研究发现，产前乙醇暴露导致大鼠胎儿中叶背核和内侧中叶核中5-羟色胺阳性神经元数量减少和迁移缺陷。在乙醇暴露的大鼠胎儿中，5-羟色胺及其代谢物（5-HIAA）的含量也降低。RT-PCR分析显示，暴露于乙醇的大鼠胎儿大脑中sonic hedgehog基因（Shh）表达减少。由于Shh与5-HT神经元的命运有关，我们得出结论，产前暴露于乙醇会损害5-HT神经元的发育，这可能导致Shh表达减少。因此，中缝中5-HT神经元的减少持续到成年，并且产前乙醇暴露大鼠表现出焦虑样行为。乙醇暴露大鼠小脑BDNF mRNA表达降低，NGF及其受体（Trk A） mRNA表达升高。海马和中隔核中OMgp和GDNF mRNA表达减少。因此，乙醇可以影响某些神经营养因子及其受体的表达。我们对新生大鼠海马来源的经乙醇处理的星形胶质细胞进行谷氨酸刺激后的细胞内钙浓度监测（第3天），发现与未经乙醇处理的星形胶质细胞相比，谷氨酸反应细胞数量减少。由于星形胶质细胞内的钙浓度可能与突触的成熟有关，结果表明乙醇可能通过星形胶质细胞内的钙信号级联影响神经回路的发育。

To identify genes and factors which are responsible for brain alterations after prenatal ethanol exposure, we examined expression of genes and determination of neurotransmitters or neurotrophic factors in ethanol-exposed rat brains. Immunohistochemical study using anti-5-HT antibody revealed that prenatal ethanol exposure resulted in a reduction in the number and defects of migration of serotonin-positive neurons in the dorsal and medial raphe nuclei of rat fetuses. In ethanol-exposed rat fetuses, amounts of both 5-HT and its metabolite (5-HIAA) were decreased as well. RT-PCR analysis revealed a reduction of sonic hedgehog (Shh) expression in the brains of ethanol-exposed rat fetuses. Because Shh is related to the fate of 5-HT neuron, we concluded that prenatal exposure to ethanol impairs the development of 5-HT neurons, and this might have resulted in the reduction of Shh expressions. Therefore, the reduction of 5-HT neurons seen in the raphe persisted into adulthood, and prenatal ethanol-exposed rats showed anxiety-like behavior. In ethanol-exposed rat cerebellum, BDNF mRNA was decreased but NGF and its receptor (Trk A) mRNA expression were increased. Also, reduction of OMgp and GDNF mRNA expression was observed in the hippocampus and the septum nucleus, respectively. Thus, it is established that ethanol can affect expression of certain neurotrophic factors and their receptors. We monitored the intracellular calcium concentration after glutamate stimulation in ethanol-treated astrocytes derived from the hippocampus of neonatal rat (Day 3), and revealed that the glutamate-reactive cell number was decreased compared to non-ethanol-treated astrocytes. Since the calcium concentration in astrocytes might be related to maturation of synapse, the result suggests that ethanol may affect development of the neural circuit via a calcium signaling cascade in astrocytes.