Gallo V, Kingsbury A, Balázs R, Jørgensen O S
J Neurosci. 1987 Jul;7(7):2203-13. doi: 10.1523/JNEUROSCI.07-07-02203.1987.
Cultures greatly enriched in granule cells from early postnatal cerebellum (P8) were grown in a medium containing fetal calf serum. Under the conditions used, nerve cells died, usually within a week, unless the K+ concentration in the medium was greater than or equal to 20 mM. The requirement for elevated [K+]e was manifested by about 3 d in vitro, and after this time continuous exposure to high [K+]e was essential for the survival of the granule cells. The initial morphological and biochemical maturation of the granule cells was similar in the presence and the absence of elevated [K+]e, suggesting that the dependence on depolarizing conditions develops in parallel with the expression of the differentiated characteristics of the cells. The positive effect of elevated [K+]e on granule cell survival was not influenced by preventing bioelectric activity in the cultures with TTX and xylocaine. On the other hand, depolarization-induced transmembrane Ca2+ flux was essential in securing the maintenance of the granule cells. Depolarized nerve cells were compromised when Ca2+ entry was blocked by elevated Mg2+, EGTA, or organic Ca2+ antagonists, while dihydropyridine Ca2+ agonists [BAY K 8644, (+)-(S)-202 79 1 and CGP 28392] were potent agents preventing nerve cell loss in the presence of 15 mM [K+]e, which was ineffective on its own. Calmodulin inhibitors (1 microM trifluoperazine or calmidazolium) blocked the beneficial effect of K+-induced depolarization on granule cells. The comparison of the timing of the differentiation and innervation of the postmitotic granule cells in vivo with the development of the K+ dependence in vitro would indicate that depolarization of the granule neurons in culture mimics the influence of the physiological stimulation in vivo through excitatory amino acid receptors, including N-methyl-D-aspartate receptors, involving Ca2+ entry and the activation of a Ca2+/calmodulin-dependent protein kinase.
从出生后早期小脑（P8）获得的富含颗粒细胞的培养物在含有胎牛血清的培养基中生长。在所用条件下，神经细胞通常在一周内死亡，除非培养基中的K⁺浓度大于或等于20 mM。对升高的[K⁺]e的需求在体外约3天时表现出来，在此之后持续暴露于高[K⁺]e对颗粒细胞的存活至关重要。在有和没有升高的[K⁺]e的情况下，颗粒细胞的初始形态和生化成熟相似，这表明对去极化条件的依赖性与细胞分化特征的表达同时发展。升高的[K⁺]e对颗粒细胞存活的积极作用不受用TTX和利多卡因阻止培养物中的生物电活动的影响。另一方面，去极化诱导的跨膜Ca²⁺通量对于确保颗粒细胞的维持至关重要。当Ca²⁺进入被升高的Mg²⁺、EGTA或有机Ca²⁺拮抗剂阻断时，去极化的神经细胞受到损害，而二氢吡啶Ca²⁺激动剂[BAY K 8644、(+)-(S)-202 79 1和CGP 28392]是在15 mM [K⁺]e存在下防止神经细胞损失的有效药物，而15 mM [K⁺]e单独使用无效。钙调蛋白抑制剂（1 μM三氟拉嗪或卡马西平）阻断了K⁺诱导的去极化对颗粒细胞的有益作用。将体内有丝分裂后颗粒细胞的分化和神经支配时间与体外K⁺依赖性的发展进行比较表明，培养物中颗粒神经元的去极化通过兴奋性氨基酸受体（包括N-甲基-D-天冬氨酸受体）模拟了体内生理刺激的影响，涉及Ca²⁺进入和Ca²⁺/钙调蛋白依赖性蛋白激酶的激活。
