A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS.
A method based on retrograde intraaxonal transport of protein for identification of cell bodies of origin of axons terminating within the CNS.
复制标题
一种基于蛋白质逆行轴突内转运的方法，用于鉴定终止于中枢神经系统内的轴突起源细胞体。
DOI：
10.1016/0006-8993(73)90016-4
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发表时间：
1973
期刊：
影响因子：
2.9
通讯作者：
Andrea Tish
中科院分区：
文献类型：
--
作者：
J. Lavail;Ken R. Winston;Andrea Tish
最近，顺行轴突内转运α 2的现象已成功地用于放射自显影鉴定中枢神经系统内的轴突投射。在Kristensson等人20和其他人18的运动神经元外周轴突实验之后，LaVail和LaVai 122证明了蛋白辣根过氧化物酶（HRP，mol.重量40，000）在小鸡的中枢神经系统内。在后一个实验中，HRP被注射到眼睛或视顶盖中，发现在细胞体内积聚，已知细胞体的轴突投射到注射部位。在本研究中，使用更小，更受限制的注射到脑实质中，我们发现，HRP的逆行轴突内运输可以用来识别那些细胞体，项目的轴突到特定区域的鸟类和成年哺乳动物中枢神经system.This研究进行了鸡，小鼠和大鼠的几个年龄。在3只1周龄雏鸡（Line 100，Cobb Hatchery，利特尔顿，马萨诸塞州）中，用Avertin 24麻醉，将HRP注射到视顶盖中。7只成年小鼠（C57 BL/6 J X CE/J F1杂交小鼠）也用Avertin麻醉，在小脑半球注射HRP：5，在上级丘注射2只。在进一步的实验中，5只用戊巴比妥麻醉的成年白化病大鼠（Fisher近交系）接受了HRP的脑内注射：3只在海马，1只在隔膜，1只在枕叶皮质。将各种体积和浓度的HRP（VI型，Sigma Chemical Co.，St. Louis）溶解在盐水中。注射后20 ~ 26小时，麻醉动物，用含1 o甲醛和1.25 o戊二醛的0.1 M磷酸盐缓冲液（24 ℃; pH7.4）固定液灌注。第二天，取出脑和在某些情况下取出脊髓，并在含有5-蔗糖的相同磷酸盐缓冲液中储存过夜。在冷冻切片机上切下40或60 μ m厚的连续切片，并在含有过氧化氢和3，3 ′-二氨基联苯胺四盐酸盐a2的培养基中孵育5-10分钟。然后将切片固定在载玻片上的明胶中，在某些情况下用0.5-甲酚紫轻微复染。
Recently the phenomenon of anterograde intraaxonal transportal, a2 has successfully been utilized for the autoradiographic identification of axonal projections within the central nervous systemL Following the experiments on peripheral axons of motor neurons by Kristensson et al. 2o and others 18, at, LaVail and LaVai122 demonstrated retrograde intraaxonal transport of the protein horseradish peroxidase (HRP, mol. wt. 40,000) within the central nervous system of the chick. In the latter experiments HRP injected into either the eye or the optic tectum was found to accumulate within cell bodies whose axons are known to project to the site of the injection. In the present study, using smaller, more restricted injections into brain parenchyma, we have found that retrograde intraaxonal transport of HRP can serve to identify those cell bodies that project their axons to specific regions of the avian and adult mammalian central nervous system.This study was performed using chicks, mice and rats of several ages. In 3 oneweek-old chicks (Line 100, Cobb Hatchery, Littleton, Mass.) anesthetized with Avertin 24, HRP was injected into the optic tectum. Seven adult mice (C57BL/6J X CE/J F1 hybrids) also anesthetized with Avertin received an injection of HRP: 5 in the cerebellar hemisphere, and two in the superior colliculus. In further experiments, 5 adult albino rats (Fisher inbred) anesthetized with Nembutal received intracerebral injections of HRP: 3 in the hippocampus, one in the septum and one in the occipital cortex. Various volumes and concentrations of HRP (Type VI, Sigma Chemical Co., St. Louis) dissolved in saline were injected. Twenty to 26 h after injection the animals were anesthetized and perfused with a fixative containing 1 o formaldehyde and 1.25~ o glutaraldehydO 6 in 0.1 M phosphate buffer (24 C; pH 7.4). The following day, the brains and in some cases the spinal cords were removed and stored overnight in the same phosphate buffer containing 5~ sucrose. Serial sections 40 or 60# m thick were cut on a freezing microtome and were incubated for 5-10 min in a medium containing hydrogen peroxide and 3, 3'-diamino-benzidine tetrahydrochloride a2. The sections were then mounted in gelatin on slides and in some cases lightly counterstained with 0.5~ cresyl violet.