Department of Pharmacology, College of Pharmacy, Seoul National University, Seoul, Korea.
J Biomed Sci. 2010 Nov 12;17(1):85. doi: 10.1186/1423-0127-17-85.
Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models.
Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis.
Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67.
These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.
孕期乙醇暴露会导致一系列精神和身体障碍，称为胎儿酒精谱系障碍（FASD）。中枢神经系统是受 FASD 影响的主要器官，神经系统症状包括智力迟钝、学习异常、儿童时期的多动和易发性癫痫以及小头畸形。在这项研究中，我们使用原代神经祖细胞（NPC）培养物和体内 FASD 模型，研究了乙醇暴露是否会对 NPC 的增殖产生不利影响，并调节谷氨酸能和 GABA 能神经元分化的正常比例。
从小鼠胚胎大脑中培养出神经祖细胞。将怀孕的小鼠和大鼠用生理盐水稀释的乙醇（2 或 4 g/kg/天）处理，从 E7 到 E16 进行体内 FASD 动物模型。通过 Western blot 分析和免疫细胞化学检测来研究蛋白质的表达水平。MTT 用于细胞活力测定。通过 BrdU 掺入、免疫细胞化学和 FACS 分析鉴定 NPC 的增殖活性。
BrdU 掺入、免疫细胞化学和 FACS 分析显示，乙醇降低了 NPC 的增殖能力。此外，乙醇通过短暂增加 Pax6、Ngn2 和 NeuroD 的表达，同时降低 Mash1 的表达，诱导谷氨酸能和 GABA 能神经元分化失衡。在体内 FASD 模型中也观察到这些转录因子的表达模式相似，以及 PSD-95 的表达增加和 GAD67 的表达减少。
这些结果表明，乙醇通过 Pax6 途径诱导谷氨酸能神经元过度分化，这可能是 FASD 患者多动或易发性癫痫等过度兴奋表型的基础。
