Gu Ya-Nan, Lee Eun-Shil, Jeon Chang-Jin
Department of Biology, School of Life Sciences, BK 21 Plus KNU Creative BioResearch Group, College of Natural Sciences, and Brain Science and Engineering Institute, Kyungpook National University, Deagu, 41566, South Korea.
Department of Biology, School of Life Sciences, BK 21 Plus KNU Creative BioResearch Group, College of Natural Sciences, and Brain Science and Engineering Institute, Kyungpook National University, Deagu, 41566, South Korea.
Exp Eye Res. 2016 Apr;145:327-336. doi: 10.1016/j.exer.2016.02.001. Epub 2016 Feb 10.
Single-cell injection after immunocytochemistry is a reliable technique for classifying neurons by their morphological structure and their expression of a particular protein. The aim of the present study was to classify the morphological types of calbindin D28k-immunoreactive retinal ganglion cells in the mouse using single-cell injection after immunocytochemistry, to estimate the density of calbindin D28k-immunoreactive retinal ganglion cells in the mouse retina. Calbindin D28k is an important calcium-binding protein that is widely expressed in the central nervous system. Calbindin D28k-immunoreactive retinal ganglion cells were identified by immunocytochemistry and then iontophoretically injected with the lipophilic dye, DiI. Subsequently, the injected cells were imaged by confocal microscopy to classify calbindin D28k-immunoreactive retinal ganglion cells based on their dendritic ramification depth within the inner plexiform layer, field size, and morphology. The cells were heterogeneous in morphology: monostratified or bistratified, with small to large dendritic field size and sparse to dense dendritic arbors. At least 10 different morphological types (CB1-CB10) of calbindin D28k-immunoreactive retinal ganglion cells were found in the mouse retina. The density of each cell type was quite variable (1.98-23.76%). The density of calbindin D28k-immunoreactive cells in the ganglion cell layer of the mouse retina was 562 cells/mm(2), 8.18% of calbindin D28k-immunoreactive cells were axon-less displaced amacrine cells, 91.82% were retinal ganglion cells, and approximately 18.17% of mouse retinal ganglion cells expressed calbindin D28k. The selective expression of calbindin D28k in cells with different morphologies may provide important data for further physiological studies of the mouse retina.
免疫细胞化学后进行单细胞注射是一种通过神经元形态结构及其特定蛋白表达来对神经元进行分类的可靠技术。本研究的目的是利用免疫细胞化学后单细胞注射的方法对小鼠中钙结合蛋白D28k免疫反应性视网膜神经节细胞的形态类型进行分类，以估计小鼠视网膜中钙结合蛋白D28k免疫反应性视网膜神经节细胞的密度。钙结合蛋白D28k是一种重要的钙结合蛋白，在中枢神经系统中广泛表达。通过免疫细胞化学鉴定出钙结合蛋白D28k免疫反应性视网膜神经节细胞，然后通过离子电泳注射亲脂性染料DiI。随后，利用共聚焦显微镜对注射细胞进行成像，根据其在内网状层内的树突分支深度、视野大小和形态对钙结合蛋白D28k免疫反应性视网膜神经节细胞进行分类。这些细胞在形态上具有异质性：单分层或双分层，树突野大小从小到大都有，树突分支从稀疏到密集。在小鼠视网膜中发现了至少10种不同形态类型（CB1 - CB10）的钙结合蛋白D28k免疫反应性视网膜神经节细胞。每种细胞类型的密度变化很大（1.98 - 23.76%）。小鼠视网膜神经节细胞层中钙结合蛋白D28k免疫反应性细胞的密度为562个细胞/mm²，8.18%的钙结合蛋白D28k免疫反应性细胞是无轴突移位无长突细胞，91.82%是视网膜神经节细胞，约18.17%的小鼠视网膜神经节细胞表达钙结合蛋白D28k。钙结合蛋白D28k在不同形态细胞中的选择性表达可能为小鼠视网膜的进一步生理学研究提供重要数据。
