Svendsen C N, Kew J N, Staley K, Sofroniew M V
Department of Anatomy, University of Cambridge, England.
J Neurosci. 1994 Jan;14(1):75-87. doi: 10.1523/JNEUROSCI.14-01-00075.1994.
Fetal septal neurons were grown in vitro under glass coverslips. This sandwich culture method significantly increased general neuronal survival, reduced glial proliferation, and permitted the removal of serum from the growth medium after 5 d in vitro. Thereafter, a simple, and completely defined, medium was used, and the effects of NGF, NGF withdrawal, and protein synthesis inhibition were examined on septal cholinergic neurons. NGF added to septal cultures at the time of plating resulted in a threefold increase in the number of cholinergic neurons seen at 14 d in vitro but had no effect on the survival of non-cholinergic cells. Cholinergic neurons identified by staining for AChE, ChAT, and p75NGFR could be maintained in serum-free, NGF-supplemented medium for over 40 d. When NGF was removed and NGF antibodies added to 14-d-old cultures, less than 30% of cholinergic neurons survived a further 4 d, but when NGF was similarly withdrawn from 35-d-old cultures, over 75% of cholinergic neurons survived. Reapplication of NGF after 3 but not after 12 or more hours of NGF withdrawal from 14-d-old cultures prevented the death of most cholinergic neurons. When NGF was withdrawn from 14-d-old cultures in the presence of the protein synthesis inhibitor cycloheximide, over 75% of the cholinergic neurons survived. These findings suggest that septal cholinergic neurons are dependent on NGF for survival only during a critical period of development and that growth factor-regulated developmental cell death may occur in CNS neurons by activation of programmed cell death requiring protein synthesis.
胎儿隔区神经元在玻璃盖玻片下进行体外培养。这种夹心培养方法显著提高了神经元的总体存活率，减少了胶质细胞的增殖，并允许在体外培养5天后从生长培养基中去除血清。此后，使用一种简单且完全确定的培养基，并研究了神经生长因子（NGF）、去除NGF以及蛋白质合成抑制对隔区胆碱能神经元的影响。在接种时向隔区培养物中添加NGF，导致体外培养14天时观察到的胆碱能神经元数量增加了两倍，但对非胆碱能细胞的存活没有影响。通过乙酰胆碱酯酶（AChE）、胆碱乙酰转移酶（ChAT）和p75神经营养因子受体（p75NGFR）染色鉴定的胆碱能神经元可以在无血清、补充NGF的培养基中维持超过40天。当去除NGF并向14天龄的培养物中添加NGF抗体时，不到30% 的胆碱能神经元在接下来的4天内存活，但当从35天龄的培养物中同样去除NGF时，超过75% 的胆碱能神经元存活。在从14天龄的培养物中去除NGF 3小时后（而非12小时或更长时间后）重新添加NGF，可防止大多数胆碱能神经元死亡。当在蛋白质合成抑制剂环己酰亚胺存在的情况下从14天龄的培养物中去除NGF时，超过75% 的胆碱能神经元存活。这些发现表明，隔区胆碱能神经元仅在发育的关键时期依赖NGF存活，并且生长因子调节的发育性细胞死亡可能通过激活需要蛋白质合成的程序性细胞死亡而在中枢神经系统神经元中发生。
