Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections
Light microscopic identification and immunocytochemical characterization of glutamatergic synapses in brain sections
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DOI：
10.1002/cne.20743
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发表时间：
2005-11-28
影响因子：
2.5
通讯作者：
Weinberg, RJ
中科院分区：
文献类型：
--
作者：
Melone, M;Burette, A;Weinberg, RJ
突触前蛋白很容易通过光镜免疫细胞化学鉴定，但在脑切片中突触后蛋白的免疫检测证明是困难的。我们对N-甲基-D-天冬氨酸受体（NMDAR）的NR 1亚基和囊泡谷氨酸转运蛋白1（VGLUT 1）进行了免疫荧光双标记。在用4%多聚甲醛固定的材料中，体感皮层的NMDAR染色仅限于切片表面，而突触前染色延伸到组织更深处。突触后蛋白染色增强弱固定材料和胃蛋白酶处理的组织，如前所述，但组织质量受损。染色也显着增强，而不损害组织质量，通过治疗灌注过程中的蛋白酶和泛素/蛋白酶体系统的抑制剂的混合物。我们对共聚焦图像进行了定量分析，以研究免疫染色如何随组织深度而变化。几乎所有的斑点免疫阳性VGLUT 1共定位与突触体素斑点，这些突触前斑点是最多的1-2 μ m下的部分表面。相反，斑点免疫阳性的NR 1亚基是最多的表面，斑点免疫阳性的NR 2亚基，SynGAP，和CaMKII。所有突触后蛋白质，但不是突触前标记物的点状染色，大大增强与抗蛋白水解剂预处理的材料。在这种材料中，与VGLUT 1相关的表面上的大多数NR 1阳性斑点可能代表突触接触。大约85%的VGLUT 1阳性斑点在SI的II-III层与NR 1阳性斑点，和类似的80%与NR 2，SynGAP，和CaMKII。这种方法可以允许系统的化学分析与光显微镜免疫细胞化学的突触。
Presynaptic proteins are readily identified by light microscopic immunocytochemistry, but immunodetection of postsynaptic proteins in brain sections proves difficult. We performed immunofluorescent double labeling for the NR1 subunit of the N-methyl-D-aspartate receptor (NMDAR) and the vesicular glutamate transporter 1 (VGLUT1). In material fixed with 4% paraformaldehyde, NMDAR staining in somatosensory cortex was restricted to the section surface, whereas presynaptic staining extended deeper into the tissue. Staining for postsynaptic proteins was enhanced in weakly fixed material and in tissue treated with pepsin, as previously reported, but tissue quality was impaired. Staining was also markedly enhanced, and without impairment of tissue quality, by treatment during perfusion with a mixture of inhibitors of proteases and the ubiquitin/proteosome system. We performed quantitative analysis of confocal images to study how immunostaining varies with depth into the tissue. Virtually all puncta immunopositive for VGLUT1 colocalized with synaptophysin puncta; these presynaptic puncta were most numerous 1-2 mu m beneath the section surface. In contrast, puncta immunopositive for the NR1 subunit were most numerous at the surface, as were puncta immunopositive for the NR2 subunit, SynGAP, and CaMKII. Punctate staining for all postsynaptic proteins, but not presynaptic markers, was substantially enhanced in material pretreated with antiproteolytic agents. The large majority of NR1-positive puncta at the surface associated with VGLUT1 in this material are likely to represent synaptic contacts. Approximately eighty-five percent of VGLUT1-positive puncta in layers II-III of SI are associated with NR1-positive puncta, and similar to 80% are associated with NR2, SynGAP, and CaMKII This approach may permit systematic analysis of the chemistry of glutamatergic synapses with light microscopic immunocytochemistry.