Johnson S M, Bywood P T
School of Medicine, Flinders University of South Australia, Adelaide 5001, Australia.
Exp Neurol. 1998 Jun;151(2):221-8. doi: 10.1006/exnr.1998.6782.
Although catecholamine neurons are vulnerable targets for neurotoxins and degenerative disease, few in vitro studies have investigated the mechanisms of neurodegeneration in these cells. We therefore developed a brain slice preparation for this purpose. Rats were killed by cervical dislocation and 400-microm-thick horizontal slices containing midbrain catecholamine neurons were incubated for 2 h in the presence or absence of kainic acid (KA, 50 microM). After fixation, the slices were recut by a technique that provided thin (40 microm) sections in the same plane as the parent slice. Catecholamine neurons in these coplanar sections were labeled by immunostaining for tyrosine hydroxylase (TH) coupled with diaminobenzidine. The topographical organization of the horizontal plane of the brain was retained in the coplanar sections, enabling precise identification of catecholamine neurons in the thin sections, by reference to an atlas in the horizontal plane. In this study we examined neurons in the substantia nigra (SN). A key feature of the immunostaining was that it revealed both the cell body and also the extensive dendritic projections of SN neurons in the horizontal plane. After treatment with KA, cell bodies remained intact but the dendrites were truncated or fragmented. The loss of dendrites is a sensitive and readily quantifiable indicator of damage. KA caused significant reductions in the proportion of SN neurons with intact dendrites and in the total length of the dendrites, measured using a computer program. The sensitive index of damage and the facility to clearly distinguish catecholamine groups that are topographically close yet functionally distinct are the principal features of the experimental approach that we have developed. The preparation offers major advantages for investigating the selective vulnerability or resistance of particular types of catecholamine neurons to damage.
尽管儿茶酚胺能神经元是神经毒素和退行性疾病的易损靶点，但很少有体外研究探讨这些细胞中神经退行性变的机制。因此，我们为此目的开发了一种脑片制备方法。通过颈椎脱臼处死大鼠，将含有中脑儿茶酚胺能神经元的400微米厚的水平切片在有无 kainic acid（KA，50 microM）的情况下孵育2小时。固定后，通过一种技术对切片进行再次切割，该技术可在与母切片相同的平面上提供薄（40微米）切片。通过酪氨酸羟化酶（TH）免疫染色结合二氨基联苯胺对这些共面切片中的儿茶酚胺能神经元进行标记。脑水平面的拓扑结构在共面切片中得以保留，通过参考水平面图谱能够精确识别薄切片中的儿茶酚胺能神经元。在本研究中，我们检查了黑质（SN）中的神经元。免疫染色的一个关键特征是它揭示了SN神经元在水平面中的细胞体以及广泛的树突投射。用KA处理后，细胞体保持完整，但树突被截断或碎片化。树突的丧失是损伤的一个敏感且易于量化的指标。使用计算机程序测量，KA导致具有完整树突的SN神经元比例以及树突总长度显著降低。损伤的敏感指标以及能够清晰区分在拓扑上接近但功能不同的儿茶酚胺能神经元群体的便利性是我们所开发实验方法的主要特征。该制备方法为研究特定类型儿茶酚胺能神经元对损伤的选择性易损性或抗性提供了主要优势。
