Yoneyama Masanori, Kitayama Tomoya, Taniura Hideo, Yoneda Yukio
Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Japan.
J Neurosci Res. 2003 Aug 1;73(3):416-26. doi: 10.1002/jnr.10622.
Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.
免疫印迹分析揭示了 N-甲基-D-天冬氨酸（NMDA）受体亚基 GluR ζ-1、GluR ε-1 和 GluR ε-2 在不同小鼠脑结构膜制剂中的异源分布情况，在小脑制剂中仅检测到 GluR ε-3 亚基。将小鼠麻醉并用 4%多聚甲醛（PA）、赞博尼溶液或卡诺伊溶液灌注，以便随后对这些 NMDA 受体亚基进行免疫组织化学检测。在灌注 4% PA 和赞博尼溶液的动物的冠状脑切片中，仅在锥体细胞层和齿状颗粒层中检测到 GluR ζ-1、GluR ε-1、GluR ε-2 和 GluR ε-3 亚基的高免疫反应性，而 GluR ε-3 亚基在这些区域本应不存在。相比之下，当用卡诺伊溶液进行浸没法固定从断头小鼠脑部分离的组织后制备切片时，在 CA1 亚区的 Oriens 层和放射层中检测到 GluR ζ-1、GluR ε-1 和 GluR ε-2 亚基的高免疫反应性，但未检测到 GluR ε-3 亚基，并且在锥体细胞层和颗粒层未检测到免疫反应性。在这些切片上，在 CA1 区的分子层、CA3 区的 Oriens 层、放射层和分子层以及齿状回的分子层中也分别发现 GluR ζ-1、GluR ε-1 和 GluR ε-2 亚基具有相对较高的免疫反应性。全身性给予海藻酸导致在用卡诺伊溶液进行浸没法固定制备的脑切片的 CA1 和 CA3 亚区中，GluR ζ-1、GluR ε-
