Zink Mathias, Schmitt Andrea, Vengeliene Valentina, Henn Fritz A, Spanagel Rainer
Central Institute of Mental Health, P.O. Box 12 21 20, D-68072 Mannheim, Germany.
Alcohol Clin Exp Res. 2004 Nov;28(11):1752-7. doi: 10.1097/01.alc.0000145810.12545.b3.
Exposure of the developing brain to ethanol disposes the fetus to fetal alcohol syndrome and causes a number of changes in several neurochemical systems. In particular, the glutamatergic system is affected by ethanol. Thus, increased sensitivity of glutamate receptors and enhanced transmembrane transport of glutamate were found in primary astrocyte cultures. However, in these in vitro studies, changes in the expression of glutamate transporters were not detected. To further characterize the influence of chronic ethanol exposure on the developing brain, we assessed the transcriptional and translational regulation of glutamate transporters in a less artificial in vitro system.
We exposed postnatal rat cortical organotypic slice cultures to ethanol (100 mM) for 4 and 8 days. Expression of the excitatory amino acid transporters EAAT1 and EAAT2 was analyzed in comparison to untreated controls with semiquantitative reverse transcriptase-polymerase chain reaction. In addition, changes in protein expression were detected by Western blotting.
In ethanol-exposed cortical slices, we observed more prominent fiber outgrowth and significantly increased gene expression (EAAT1: +35%, p = 0.029; EAAT2: +251%, p = 0.015). These findings could be verified on the protein level, because Western blot analysis revealed significantly higher levels of EAAT1 (+76%; p = 0.008) and EAAT2 (+104%; p = 0.018) in ethanol-treated slices compared with controls.
Our results are in concert with earlier studies describing the induction of glutamate transport by ethanol. Enhanced expression of EAAT1 and EAAT2 after ethanol exposure can be viewed as a maladaptive process that disposes the developing brain to fetal alcohol syndrome.
发育中的大脑暴露于乙醇会使胎儿易患胎儿酒精综合征，并导致多个神经化学系统发生一系列变化。特别是，谷氨酸能系统会受到乙醇的影响。因此，在原代星形胶质细胞培养物中发现谷氨酸受体的敏感性增加以及谷氨酸的跨膜转运增强。然而，在这些体外研究中，未检测到谷氨酸转运体表达的变化。为了进一步表征慢性乙醇暴露对发育中大脑的影响，我们在一个较少人为因素的体外系统中评估了谷氨酸转运体的转录和翻译调控。
我们将出生后大鼠皮质器官型切片培养物暴露于乙醇（100 mM）中4天和8天。与未处理的对照相比，通过半定量逆转录 - 聚合酶链反应分析兴奋性氨基酸转运体EAAT1和EAAT2的表达。此外，通过蛋白质印迹检测蛋白质表达的变化。
在暴露于乙醇的皮质切片中，我们观察到更明显的纤维生长和基因表达显著增加（EAAT1：+35%，p = 0.029；EAAT2：+251%，p = 0.015）。这些发现可以在蛋白质水平上得到验证，因为蛋白质印迹分析显示，与对照相比，乙醇处理切片中EAAT1（+76%；p = 0.008）和EAAT2（+104%；p = 0.018）的水平显著更高。
我们的结果与早期描述乙醇诱导谷氨酸转运的研究一致。乙醇暴露后EAAT1和EAAT2表达的增强可被视为一种适应不良的过程，使发育中的大脑易患胎儿酒精综合征。
