Weiss S, Pin J P, Sebben M, Kemp D E, Sladeczek F, Gabrion J, Bockaert J
Proc Natl Acad Sci U S A. 1986 Apr;83(7):2238-42. doi: 10.1073/pnas.83.7.2238.
Striatal neurons were cultured from the fetal mouse brain and maintained in serum-free medium for 14-21 days in vitro (DIV). Pretreatment of the culture dishes successively with a polycation followed by fetal calf serum resulted in rapid neuron attachment and neurite proliferation. After 9-10 DIV, electron microscope observations revealed the presence of vesicles in axon terminals forming mature synapses with axons and perikarya of adjacent neurons and in varicosities along extended axons. Synapsin I, a synaptic vesicle-specific protein, was present only in neuronal perikarya after 3 DIV, in perikarya and in varicosities along extended axons after 6 DIV, and in varicosities and contact points between axon terminals and adjacent axons or perikarya after 11-14 DIV. Neurotransmitter-stimulated intracellular formation of cAMP decreased markedly during neuronal differentiation. Inositol phosphate formation in response to neurotransmitters, however, increased significantly throughout the period of striatal neuronal development. K+ (56 mM) depolarization resulted in a 2-fold increase in endogenous gamma-aminobutyric acid (GABA) release from striatal neurons, 50% of which was Ca2+-dependent, between 3 and 11 DIV. Between 11 and 14 DIV, subsequent to synapse formation (as revealed by electron microscope observations), GABA release evoked by 56 mM K+ increased up to 5-fold, 75% of which was Ca2+-dependent. It appears that the complete differentiation of striatal neurons in serum-free medium may provide a suitable model for the study of the physiological and regulatory mechanisms involved in nerve cell development.
从胎鼠脑中培养纹状体神经元，并在无血清培养基中体外培养14 - 21天（体外培养天数，DIV）。培养皿先用聚阳离子预处理，然后用胎牛血清处理，可使神经元迅速附着并促进神经突增殖。体外培养9 - 10天后，电子显微镜观察显示轴突终末存在囊泡，这些轴突终末与相邻神经元的轴突和胞体形成成熟突触，并且在延伸轴突上的膨体中也有囊泡。突触素I是一种突触囊泡特异性蛋白，在体外培养3天后仅存在于神经元胞体中，6天后存在于胞体和延伸轴突上的膨体中，11 - 14天后存在于轴突终末与相邻轴突或胞体之间的膨体和接触点中。在神经元分化过程中，神经递质刺激的细胞内cAMP形成显著减少。然而，在整个纹状体神经元发育期间，对神经递质作出反应的肌醇磷酸形成显著增加。在体外培养3至11天期间，56 mM K⁺去极化导致纹状体神经元内源性γ-氨基丁酸（GABA）释放增加2倍，其中50%依赖于Ca²⁺。在体外培养11至14天期间，在突触形成后（如电子显微镜观察所示），56 mM K⁺诱发的GABA释放增加至5倍，其中75%依赖于Ca²⁺。似乎在无血清培养基中纹状体神经元的完全分化可能为研究神经细胞发育所涉及的生理和调节机制提供一个合适的模型。
