一种获得小鼠背侧齿状回健康颗粒细胞的脑片制备方法

A method for getting health granule cells in dorsal dentate gyrus from brain slice of mice

目的：研究获得背侧齿状回健康颗粒细胞的制片方法。方法：成年小鼠。（1）控制切片角度,尽量保留颗粒细胞顶树突的完整沿水平20～30°切片角度获取海马横向（transverse）脑片;（2）注意区分脑片的正、反面选择海马脑片反面的背侧齿状回上片;（3）合并使用其它保护脑片细胞活性的措施,如采用无钠切片液,切片液中添加抗氧化剂等。结果：本方法所得特定角度的海马横向脑片,其上有大量颗粒细胞表现出轮廓清晰、柔和、胞体圆润的健康状态,且记录出颗粒细胞兴奋性微小突触后电流（mEPSCs）的频率和幅度,均较传统海马横向脑片方法有明显增加。结论：适当的切片角度可保持颗粒细胞远端树突末梢的完整,提高细胞的活性,获得大量健康颗粒细胞。提示在制备脑片时要考虑感兴趣细胞整体的走行,保证切片时细胞整体的完整性。

Objective：To establish a protocol for keeping the viability of granule cells in dorsal dentate gyrus（dDG）from brain slice.Methods：The adult mice were used for the experiment.（1） The hippocampal transverse slices were cut with horizontal 20-30° angle for keeping integrity of the apical dendrite of granule cells;（2） The obverse and reverse side of the slices need be differentiated by the experimenter.The granule cells in the suprapyramidal blade of reverse side of the slices were selected and recorded with whole cell patch clamping;（3） Other methods including zero sodium cutting solution with antioxidant were used to keep slice health.Results：The transverse hippocampal slices with this protocol have plenty of healthy granule cells which showed typical elliptical cell body and cleaning cell surface.The miniature excitatory postsynaptic currents（mEPSCs） in granule cells with this protocol showed higher frequency and bigger amplitude than traditional transverse slice protocol.Conclusion：This protocol can increase the viability of granule cells in dDG through keeping the apical dendritic tree intact.It suggests that the whole cell process and 3D structure are the issue which we need think about during cutting brain slice.