Martinic M, Lambert M P, Hua S, Klein W L
Department of Neurobiology and Physiology, Northwestern University, Evanston, Illinois 60208.
J Neurobiol. 1992 Apr;23(3):252-69. doi: 10.1002/neu.480230305.
To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.
为研究中枢神经系统（CNS）胆碱能发育的早期事件，将来自大鼠基底前脑组织的细胞在体内神经发生仍活跃的年龄（胚胎第15天，E15）进行培养。这些细胞在限定培养基中迅速死亡，5 - 10小时后死亡率达50%，但嗅球（基底前脑靶组织）的可溶性蛋白可完全阻止这种死亡，这扩展了早期的观察结果（兰伯特、梅杰里安、加登和克莱因，1988年）。经处理的培养物能够将胸苷掺入DNA，且大多数掺入3H - 胸苷的细胞（超过90%）神经丝染色也呈阳性，证实了补充培养基中神经元的增殖。一小部分3H - 胸苷标记的细胞胶质纤维酸性蛋白（GFAP）呈阳性，但支持星形胶质细胞增殖的生长因子[表皮生长因子（EGF）、碱性成纤维细胞生长因子（bFGF）和胰岛素样生长因子（IGF - 1）]不足以支持神经元生长。在补充培养基培养5天后，近50%的细胞显示胆碱乙酰转移酶（ChAT）免疫荧光。胆碱能神经元通常形成与非胆碱能细胞分开的簇。如果用阿非迪霉素处理年轻培养物以阻断DNA合成，这些成熟培养物就不会发育。数据表明，非常年幼的大鼠基底前脑细胞培养物可具有神经源性，产生大量胆碱能神经元，且早期细胞增殖对长期培养存活至关重要。
