The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice
The effects of cutting solutions on the viability of GABAergic interneurons in cerebral cortical slices of adult mice
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DOI：
10.1016/j.jneumeth.2008.02.021
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发表时间：
2008-06-15
影响因子：
3
通讯作者：
Kaneko, Takeshi
中科院分区：
文献类型：
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作者：
Tanaka, Yasuyo;Tanaka, Yasuhiro;Kaneko, Takeshi
为了在成年小鼠大脑皮层切片中获得有活力的gaba能中间神经元，我们研究了切片切割溶液对GAD67-GFP敲入小鼠表达绿色荧光蛋白（GFP）的皮层神经元活力的影响。在孵育的切片中，几乎没有GFP阳性神经元的细胞核被碘化丙啶标记，这表明GFP荧光是gaba能中间神经元活力的有用指标。几种切割溶液与盐基溶液比较，n-甲基- d -氨基葡萄糖无钠溶液最能使gfp阳性神经元数量保持在灌注固定脑水平附近。与盐水溶液相比，无钠溶液切片在切后1 ~ 6 h、切后30 ~ 60 μ m深度的皮层v ~ vi层中gfp阳性神经元数量较多。此外，在高钙浓度（5 mM）或高温（37℃）的切割条件下，GFP阳性神经元数量减少，在0℃的切割条件下，GFP荧光减少。本研究结果表明，在20℃的无钠溶液中切割大脑是制备gabp能中间神经元存活的皮质切片的方法。该方法可用于成人大脑皮层中间神经元切片的电生理和形态学研究。(c) 2008 Elsevier B.V.版权所有
To obtain viable GABAergic interneurons in cerebral cortical slices of adult mice, we investigated the effects of slice cutting solutions on the viability of green fluorescent protein (GFP)-expressing cortical neurons in GAD67-GFP knock-in mice. Almost no nuclei of GFP-positive neurons were labeled with propidium iodide in incubated slices, suggesting that GFP fluorescence was a useful indicator for the viability of GABAergic interneurons. When several cutting solutions were compared with saline-based solution, N-methyl-D-glucamine-based sodium-free solution was most effective to keep the number of GFP-positive neurons near the level of perfusion-fixed brain. GFP-positive neurons in slices cut with sodium-free solution were more numerous in cortical layers V-VI, at 30-60 mu m depth from the cut surface and 1-6 h after cutting than those with saline-based solution. Furthermore, the number of GFP-positive neurons decreased in the cutting condition of high calcium concentration (5 mM) or high temperature (37 degrees C), and GFP fluorescence decreased when cut at 0 degrees C. The present results indicate that cutting the brain at 20 degrees C in sodium-free solution is a method for preparing cortical slices with GABAergic interneurons viable. This method would thus be useful for electrophysiological and morphological studies of cortical interneurons in slice preparations of the adult brain. (c) 2008 Elsevier B.V. All rights reserved.