Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, 4301 W Markham Street, #522-3 Little Rock, AR 72205-7199, USA.
Neuroscience. 2010 Sep 1;169(3):1438-48. doi: 10.1016/j.neuroscience.2010.06.008. Epub 2010 Jun 11.
Ethanol exposure during postnatal days (PN) 4-6 in rats alters cerebellar development resulting in significant loss of Purkinje cells. There is little knowledge, however, on what happens to the neurons that survive. In this study, rat pups were treated with a daily dose of ethanol (either 3.6 or 4.5 g/kg body weight) delivered by intragastric intubation on PN4, PN4-6, or PN7-9. Then the interactions between climbing fibers and Purkinje cells were examined on PN14 using confocal microscopy. Mid-vermal cerebellar sections were stained with antibodies to calbindin-D28k (to visualize Purkinje cells) and vesicular glutamate transporter 2 (VGluT2, to visualize climbing fibers). Confocal z-stack images were obtained from Lobule 1 and analyzed with Imaris software to quantify the staining of the two antibodies. The VGluT2 immunostaining was significantly reduced in the PN4 and PN4-6 ethanol groups for the 4.5 g/kg dose level, compared to controls, indicating that the cerebellar circuitry was significantly altered following developmental ethanol exposure. Not only were there fewer Purkinje cells following ethanol exposure, but the surviving neurons had significantly fewer VGluT2-labeled synapses. These alterations in the synaptic integrity were both dose dependent and temporally dependent.
在大鼠出生后第 4-6 天（PN）期间暴露于乙醇会改变小脑的发育，导致浦肯野细胞大量丧失。然而，对于幸存的神经元会发生什么，人们知之甚少。在这项研究中，通过胃内插管在 PN4、PN4-6 或 PN7-9 每天给大鼠幼仔施用一剂乙醇（3.6 或 4.5 g/kg 体重）。然后，在 PN14 时使用共聚焦显微镜检查攀援纤维和浦肯野细胞之间的相互作用。用针对钙结合蛋白-D28k（用于可视化浦肯野细胞）和囊泡谷氨酸转运体 2（VGluT2，用于可视化攀援纤维）的抗体对中间蚓状叶小脑切片进行染色。从 1 叶获得共聚焦 z 堆叠图像，并使用 Imaris 软件进行分析以量化两种抗体的染色。与对照组相比，4.5 g/kg 剂量水平的 PN4 和 PN4-6 乙醇组的 VGluT2 免疫染色明显减少，这表明发育性乙醇暴露后小脑回路发生了显著改变。不仅在暴露于乙醇后浦肯野细胞数量减少，而且幸存的神经元的 VGluT2 标记突触数量也明显减少。突触完整性的这些改变既具有剂量依赖性，也具有时间依赖性。
