ESTABLISHMENT OF A LONG-TERM PRIMARY CULTURE OF STRIATAL NEURONS
ESTABLISHMENT OF A LONG-TERM PRIMARY CULTURE OF STRIATAL NEURONS
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DOI：
10.1016/0165-3806(90)90239-u
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发表时间：
1990-03-01
期刊：
影响因子：
--
通讯作者：
DUMUIS, A
中科院分区：
文献类型：
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作者：
SEBBEN, M;GABRION, J;DUMUIS, A
本文描述了一种获得纹状体神经元长期原代培养物（持续超过 8 周）的新方法。该方法的独创性在于：(1)在无血清培养基中开始培养3天，使神经元附着和神经突增殖，以及非神经元细胞(主要由星形胶质细胞组成)死亡。 (2)在体外3天(3 DIV)后引入有限量的胎牛血清(FCS)(2-5%)，这可能提供最佳的神经元存活和附着因子，以及有限量的星形胶质细胞增殖因子。引入血清的时间以及引入血清的量是关键因素。通过相差和透射电子显微镜，我们观察到神经元继续发育神经突延伸、突触小泡和突触形成高达 50 DIV。神经元膜和突触接触在高达 50 DIV 时特别健康。有趣的是，星形胶质细胞的数量恒定在 30-50 DIV 之间，并限制在 10% 左右。因此，我们获得了神经元和星形胶质细胞分化和增殖之间的平衡。星形胶质细胞的数量较少，加上添加的FCS的比例较低，这可能为神经元存活和分化提供了必要的因素，而高密度的分化神经元则抑制了星形胶质细胞的增殖。这些神经元培养物的明确稳定性与这里研究的药理反应的稳定性是平行的：卡巴胆碱和使君子酸受体与磷酸肌醇生产系统的偶联。这里描述的培养方法可能特别适合对神经元以及神经元和星形胶质细胞之间的相互作用进行生化药理学和生物学研究。
A new method of obtaining long-term primary cultures (lasting more than 8 weeks) of striatal neurons is described in this paper. The originality of the method consists of: (1) starting the culture for 3 days in a serum-free medium which allows attachment and neurite proliferation of neurons as well as the death of non-neuronal cells (mainly consisting of astrocytes); (2) introducing a limited amount of fetal calf serum (FCS) (2-5%) after 3 days in vitro (3 DIV), which likely provides optimal neuronal survival and attachment factors, and a limited amount of astrocyte proliferating factors. The period of introduction of serum, as well as the amount of serum introduced are critical factors. By phase contrast and transmission electron microscopy, we observed that neurons continued to develop neurite extensions, synaptic vesicles and synapse formations up to 50 DIV. Neuronal membranes, and synaptic contacts were particularly healthy up to 50 DIV. Interestingly, the number of astrocytes was constant between 30-50 DIV and limited to about 10%. We therefore obtained an equilibrium between neuronal and astrocyte differentiation and proliferation. It is likely that the small population of astrocytes, plus the low percentage of FCS added, provide essential factors for neuronal survival and differentiation, whereas a high density of differentiated neurons inhibited astrocyte cell proliferation. The clear-cut stability of these neuronal cultures goes in parallel with the stability of the pharmacological responses studied here: the coupling of carbachol and quisqualate receptors with the inositol phosphate production system. The culture method described here could be of particular interest to pursue biochemical pharmacological and biological studies on neurons as well as on reciprocal interactions between neurons and astrocytes.