Combining laser scanning confocal microscopy and electron microscopy to determine sites of synaptic contact between two identified neurons
Combining laser scanning confocal microscopy and electron microscopy to determine sites of synaptic contact between two identified neurons
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DOI：
10.1016/s0165-0270(00)00184-9
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发表时间：
2000-04-15
影响因子：
3
通讯作者：
Meyrand, P
中科院分区：
文献类型：
--
作者：
Cabirol-Pol, MJ;Mizrahi, A;Meyrand, P
在这里，我们报告了一种相关的共聚焦和电子显微镜(EM)的双重标记方法，它允许在相同的制备中选择性地表征两个单一识别的神经元之间的结构关系。利用龙虾的口胃神经系统，我们通过在一个神经元细胞内注射荧光黄(LY)，在其伴侣中注射罗丹明(RhoDamine，RdH)和辣根过氧化物酶(HRP)的混合物，对已识别的、突触相连的神经元对进行标记。首先，用激光扫描共聚焦显微镜(LSCM)观察LY和RHD染色神经元的整体，以分离可能的突触接触的神经绒毛区域。第二，常规处理后的电子显微镜(免疫金标记法显示LY，DAB显示HRP)，在EM中可见紧密对位的区域。这项技术使我们能够确定两个细胞之间所有紧密接触的区域，然后使用电子显微镜来确定这些限制区域中每个区域是否存在突触接触。这些技术使我们能够证明，几乎没有并列的区域，而且这些区域中只有极小一部分实际上是两个标记神经元之间的突触接触区域。(C)2000由爱思唯尔科学公司出版。保留所有权利。
Here we report a double labelling method for correlative confocal and electron microscopy (EM) which allows selective characterisation of structural relationships between two single identified neurons in the same preparation. Using the lobster stomatogastric nervous system, we labelled pairs of identified, synaptically-connected neurons by intracellular injection of Lucifer Yellow (LY) in one neuron and a mixture of Rhodamine (Rdh) and Horseradish Peroxidase (HRP) in its partner. First, whole-mounts of LY- and Rdh-stained neurons were visualized using laser scanning confocal microscopy (LSCM) in order to isolate neuropilar regions of possible synaptic contact. Second, after conventional treatment for electron microscopy (LY was revealed with immunogold and HRP with DAB), areas of close appositions were viewed in EM. This technique allowed us to determine all the regions of close contact between two cells, and then to use electron microscopy to determine the presence or absence of synaptic contact within each of these restricted areas. These techniques enabled us to show that there were few areas of apposition and that only an extremely small proportion of these areas was in fact regions of synaptic contact between the two labelled neurons. (C) 2000 Published by Elsevier Science B.V. All rights reserved.