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

为了确定导致出生前酒精暴露后大脑改变的基因和因素，我们检测了酒精暴露大鼠大脑中基因的表达和神经递质或神经营养因子的测定。用抗5-羟色胺抗体进行免疫组织化学研究表明，孕期酒精暴露导致胎鼠中缝背内侧核内5-羟色胺阳性神经元迁移的数量和缺陷减少。在酒精暴露的大鼠胚胎中，5-羟色胺及其代谢物(5-HIAA)的含量也减少。RT-PCR分析显示，乙醇暴露的大鼠胚胎大脑中Sonic Hedgehog(Shh)的表达减少。由于Shh与5-羟色胺神经元的命运有关，我们得出结论，产前暴露于乙醇会损害5-羟色胺神经元的发育，这可能是导致Shh表达减少的原因。因此，中缝核内5-羟色胺神经元的减少持续到成年期，出生前暴露于酒精的大鼠表现出焦虑样的行为。在乙醇暴露的大鼠小脑，BDNF基因表达降低，而NGF及其受体(Trk A)基因表达增加。此外，在海马区和隔核分别观察到OMgp和GDNFm RNA的表达减少。因此，乙醇可以影响某些神经营养因子及其受体的表达。我们监测了谷氨酸刺激后新生大鼠海马区星形胶质细胞(第3天)谷氨酸刺激后的细胞内钙离子浓度，发现谷氨酸反应细胞的数量比未酒精处理的星形胶质细胞减少。由于星形胶质细胞内的钙离子浓度可能与突触的成熟有关，这一结果提示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.