Dolleman-Van der Weel M J, Wouterlood F G, Witter M P
Graduate School Neurosciences Amsterdam, Faculty of Medicine, Department of Anatomy and Embryology, Vrije Universiteit, Netherlands.
J Neurosci Methods. 1994 Jan;51(1):9-21. doi: 10.1016/0165-0270(94)90021-3.
The simultaneous use of different neuroanatomical anterograde tracers provides a potentially powerful method to study the convergence of afferent systems in a particular brain area. However, a simple routine procedure to apply multiple anterograde tracers in conjunction with their simultaneous visualization is still missing. We report an easy and straightforward application of three sensitive anterograde tracers: Phaseolus vulgaris leucoagglutinin (PHA-L), rhodamine-conjugated dextran amine (RDA) and biotin-conjugated dextran amine (BDA). These tracers can be visualized simultaneously and permanently through a triple-staining procedure with nickel-enhanced diaminobenzidine (DAB-Ni), DAB and 1-naphthol/Azur B as chromogens. Our test model comprised the projections from the nucleus reuniens thalami and entorhinal cortex. Both projection systems show a high degree of overlap in their terminal fields in the hippocampus. Two tracers were injected in the left and right entorhinal cortex, respectively; a third tracer was injected in the nucleus reuniens. This combination of injections provided a good opportunity to compare the three tracers in one and the same animal. PHA-L, RDA and BDA, injected in either of the injection sites, turned out to be equally sensitive and revealed the morphology of the involved projection systems in great detail. The triple-staining protocol yielded an excellent, simultaneous detectability of the three tracers with a remarkably low background level. Thus, the combination of the anterograde tracers PHA-L, RDA and BDA, in conjunction with the triple-staining procedure, offers a very attractive approach for neuroanatomical research.
同时使用不同的神经解剖学顺行示踪剂为研究特定脑区传入系统的汇聚提供了一种潜在的强大方法。然而，目前仍缺乏一种简单的常规程序来同时应用多种顺行示踪剂并实现它们的同时可视化。我们报告了一种简便直接的方法，可应用三种敏感的顺行示踪剂：菜豆白细胞凝集素（PHA-L）、罗丹明偶联葡聚糖胺（RDA）和生物素偶联葡聚糖胺（BDA）。通过使用镍增强二氨基联苯胺（DAB-Ni）、DAB和1-萘酚/天青B作为显色剂的三重染色程序，可以同时且永久地可视化这些示踪剂。我们的测试模型包括丘脑 reunions 核和内嗅皮层的投射。这两个投射系统在海马体的终末场显示出高度重叠。分别在左右内嗅皮层注射两种示踪剂；在丘脑 reunions 核注射第三种示踪剂。这种注射组合为在同一动物中比较这三种示踪剂提供了一个很好的机会。注射在任何一个注射部位的PHA-L、RDA和BDA结果显示同样敏感，并能非常详细地揭示相关投射系统的形态。三重染色方案对三种示踪剂具有出色的同时可检测性，背景水平极低。因此，顺行示踪剂PHA-L、RDA和BDA与三重染色程序相结合，为神经解剖学研究提供了一种非常有吸引力的方法。
