Van Lookeren Campagne M, Dotti C G, Jap Tjoen San E R, Verkleij A J, Gispen W H, Oestreicher A B
Rudolf Magnus Institute, Division of Molecular Neurobiology, Utrecht, The Netherlands.
Neuroscience. 1992 Sep;50(1):35-52. doi: 10.1016/0306-4522(92)90380-k.
Hippocampal pyramidal neurons cultured in vitro gradually develop morphologically and biochemically distinct axons and dendrites, resulting in functional neuronal polarization [Dotti C. G. et al. (1988) J. Neurosci. 8, 1454-1468]. We have studied the distribution of the growth-associated protein B-50 in hippocampal neurons of the rat at stage 3 of development by means of light and electron microscopic immunocytochemistry. Hippocampal neurons grown for two to three days in vitro were aldehyde fixed and immunolabelled using polyclonal rabbit antibodies to B-50 and goat anti-rabbit immunoglobulins tagged with 1 nm gold particles. In order to permit visualization by both light and electron microscopy, the gold probes were silver intensified. Light microscopy demonstrated the absence of B-50 immunostaining in living neurons and the presence after permeabilization by fixation and subsequent treatment of the neurons with sodium borohydride, indicating that B-50 is located intracellularly. Both immunofluorescence and immunogold-silver labelling revealed that B-50 immunoreactivity outlined all neurites of the morphologically polarized neurons. For quantitative electron microscopy, six morphologically polarized neurons (developmental stage 3) were carefully selected from immunolabelled Epon-embedded neurons and processed completely to ultrathin sections. In this way the ultrastructural localization of B-50 has been studied in the cell body, the neurites and their growth cones. For each sectioned neuron, the relative distribution of the gold-silver deposits (representing B-50) over the plasma membrane of various cellular compartments was quantitated. B-50 is located at the plasma membrane of the neuronal cell body and all neurites including their growth cones. The density of B-50 on the plasma membrane of growth cones is not different from that of the neuritic shaft. In addition, B-50 is present on the cytosolic side of the membrane of small electron-lucent vesicles (average diameter 102.7 +/- 2.5 nm) resembling transport vesicles. These vesicles are present in the cell body and the neurites. A two-fold concentration is found in the central region of the growth cones, suggesting a role of these vesicles in axonal transport, membrane insertion and (or) recycling. Since, at the onset of neuronal polarization, B-50 is present at the plasma membrane in all compartments of the hippocampal neuron, we suggest that at this stage of development B-50 does not participate directly in the processes leading to morphological polarization.(ABSTRACT TRUNCATED AT 400 WORDS)
体外培养的海马锥体神经元在形态和生化方面逐渐发育出不同的轴突和树突，从而导致神经元功能极化[多蒂C.G.等人（1988年）《神经科学杂志》8卷，1454 - 1468页]。我们通过光镜和电镜免疫细胞化学方法研究了发育第3阶段大鼠海马神经元中生长相关蛋白B - 50的分布。将体外培养两到三天的海马神经元用醛固定，并用抗B - 50的兔多克隆抗体和标记有1纳米金颗粒的羊抗兔免疫球蛋白进行免疫标记。为了能在光镜和电镜下观察，对金探针进行银增强。光镜显示活神经元中不存在B - 50免疫染色，而在固定使其通透并随后用硼氢化钠处理神经元后出现免疫染色，这表明B - 50位于细胞内。免疫荧光和免疫金银标记均显示，B - 50免疫反应性勾勒出形态极化神经元的所有神经突。为进行定量电镜分析，从免疫标记的环氧树脂包埋神经元中仔细挑选出6个形态极化神经元（发育第3阶段）并制成超薄切片。通过这种方式研究了B - 50在细胞体、神经突及其生长锥中的超微结构定位。对于每个切片的神经元，定量分析了金银沉积物（代表B - 50）在各种细胞区室质膜上的相对分布。B - 50位于神经元细胞体和所有神经突包括其生长锥的质膜上。生长锥质膜上B - 50的密度与神经突轴的密度无差异。此外，B - 50存在于类似运输小泡的小电子透明小泡（平均直径102.7±2.5纳米）膜的胞质侧。这些小泡存在于细胞体和神经突中。在生长锥的中心区域发现其浓度增加了两倍，这表明这些小泡在轴突运输、膜插入和（或）再循环中起作用。由于在神经元极化开始时，B - 50存在于海马神经元所有区室的质膜上，我们认为在发育的这个阶段B - 50不直接参与导致形态极化的过程。（摘要截选至400字）
