Meredith G E, Wouterlood F G
Department of Anatomy and Embryology, Faculty of Medicine, Free University, Amsterdam, The Netherlands.
Microsc Res Tech. 1993 Jan 1;24(1):31-42. doi: 10.1002/jemt.1070240105.
The injection of the dye Lucifer Yellow (LY) into neurons in slices of fixed brain is used to associate cells displaying a particular dendritic geometry with a specific pattern of neuronal connectivity. In the present report we expand on this technique by combining it at the electron microscopic level with immunocytochemistry and/or degeneration for the study of synaptic relationships. As a model we use the projection neurons of nucleus accumbens. These neurons were retrogradely labeled in vivo with injections or a fluorescent tracer. Fast Blue, into the ventral mesencephalon. Using epifluorescent monitoring, these neurons were located in perfusion-fixed brain slices and intracellularly injected with LY. They were visualized in the light and electron microscope using a peroxidase-antiperoxidase immunocytochemical method. Certain afferent connections of these neurons were identified in the same tissue through the use of either dual-label immunocytochemistry or anterograde degeneration combined with a single-label immunoreaction. In the dual-label procedure, a silver-gold intensification of the diaminobenzidine (DAB) reaction product for the first antigen (LY) was contrasted with a nonintensified reaction product for the second antigen (tyrosine hydroxylase [TH]). Ultrastructurally, metallic gold particles appeared to be dispersed over the immunolabeled perikarya, dendrites, and, occasionally, axonal terminals of LY-injected neurons whereas the flocculent DAB reaction product was present in TH-containing axons and terminals. Following lesions of the ventral subiculum in the hippocampal formation, degenerating axon terminals were detected in nucleus accumbens along with immunoreacted, LY-injected neurons. The techniques outlined in this report should prove invaluable for the study of the synaptic interactions of identified neurons. They can be reliably reproduced with a high yield per experiment.
将荧光黄（LY）注入固定脑片的神经元中，用于将呈现特定树突形态的细胞与特定的神经元连接模式联系起来。在本报告中，我们通过在电子显微镜水平将其与免疫细胞化学和/或变性相结合来扩展该技术，以研究突触关系。作为模型，我们使用伏隔核的投射神经元。这些神经元在体内通过向腹侧中脑注射荧光示踪剂快蓝进行逆行标记。利用落射荧光监测，在灌注固定的脑片中定位这些神经元，并对其进行细胞内LY注射。使用过氧化物酶-抗过氧化物酶免疫细胞化学方法在光学显微镜和电子显微镜下对它们进行观察。通过使用双标记免疫细胞化学或顺行变性结合单标记免疫反应，在同一组织中鉴定出这些神经元的某些传入连接。在双标记过程中，对第一种抗原（LY）的二氨基联苯胺（DAB）反应产物进行银金强化，与第二种抗原（酪氨酸羟化酶[TH]）的未强化反应产物形成对比。在超微结构上，金属金颗粒似乎分散在LY注射神经元的免疫标记核周体、树突以及偶尔的轴突终末上，而絮状的DAB反应产物则存在于含TH的轴突和终末中。在海马结构腹侧下托损伤后，在伏隔核中检测到变性的轴突终末以及免疫反应的、LY注射的神经元。本报告中概述的技术对于研究已鉴定神经元的突触相互作用应具有极高的价值。它们可以在每个实验中以高产量可靠地重现。
