Messing A, Kim S U
Brain Res. 1981 Mar 16;208(2):479-86. doi: 10.1016/0006-8993(81)90581-3.
We have maintained embryonic chick ciliary ganglion neurons in dissociated cell culture and studied the progressive appearance of surface receptors for [125I]alpha-bungarotoxin. Cultures were established from 8-day-old embryos and fed a medium supplemented with 180 micrograms/ml of a soluble protein extract prepared from the eye, the target organ for the ciliary ganglion. Approximately 8064 neurons survived per ganglion and there was no evident loss of neurons through two weeks in culture. Binding of [125I]alpha-bungarotoxin was determined at room temperature in intact cells still attached to their coverslips. Non-specific binding was less than 2% of the total. Specific binding of [125I]alpha-bungarotoxin was saturable with respect to both time of incubation (20-30 min) and concentration of toxin (5-10 nM), with an apparent Kd = 1.0 nM. Binding sites for [125I]alpha-bungarotoxin increased during the first week in culture from 1.8 fmol per 10(4) neurons at 1 day in vitro (DIV) to 8.6 fmol per 10(4) neurons at 7 DIV, after which the number of sites seemed to plateau. Light microscopic autoradiography was performed on cultures at 4 DIV and showed most of the grains associated with the surfaces of neuronal cell bodies, while scattered grains occurred over neuronal processes. When compared with previous reports on the in vivo development of alpha-bungarotoxin receptors in chick ciliary ganglia, the appearance of receptors in these cultured neurons followed a time course similar to, but at lower levels, than, their in vivo counterparts. Nevertheless, this culture system should prove useful for the study of questions concerning the regulation, surface distribution and intracellular pathways of neuronal alpha-bungarotoxin receptors.
我们将鸡胚睫状神经节神经元维持在解离细胞培养中，并研究了[125I]α-银环蛇毒素表面受体的逐步出现情况。培养物取自8日龄胚胎，并用补充了180微克/毫升由睫状神经节的靶器官眼睛制备的可溶性蛋白提取物的培养基喂养。每个神经节约有8064个神经元存活，在培养的两周内神经元没有明显损失。在室温下，对仍附着在盖玻片上的完整细胞测定[125I]α-银环蛇毒素的结合情况。非特异性结合小于总量的2%。[125I]α-银环蛇毒素的特异性结合在孵育时间（20 - 30分钟）和毒素浓度（5 - 10 nM）方面都是可饱和的，表观解离常数Kd = 1.0 nM。培养第一周期间，[125I]α-银环蛇毒素的结合位点从体外培养第1天（DIV）时每10(4)个神经元1.8飞摩尔增加到第7 DIV时每10(4)个神经元8.6飞摩尔，此后位点数量似乎趋于稳定。在4 DIV时对培养物进行光镜放射自显影，结果显示大多数银粒与神经元细胞体表面相关，而在神经突起上有散在的银粒。与先前关于鸡睫状神经节中α-银环蛇毒素受体体内发育的报道相比，这些培养神经元中受体的出现时间进程与体内对应物相似，但水平较低。然而，这种培养系统对于研究有关神经元α-银环蛇毒素受体的调节、表面分布和细胞内途径的问题应该是有用的。
