Characterization of Calcium Responses and Electrical Activity in Differentiating Mouse Neural Progenitor Cells In Vitro
Characterization of Calcium Responses and Electrical Activity in Differentiating Mouse Neural Progenitor Cells In Vitro
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DOI：
10.1093/toxsci/kft261
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发表时间：
2014-02-01
影响因子：
3.8
通讯作者：
Westerink, Remco H. S.
中科院分区：
文献类型：
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作者：
de Groot, Martje W. G. D. M.;Dingemans, Milou M. L.;Westerink, Remco H. S.
发育神经毒性（DNT）测试的体外方法有可能减少动物使用，并增加对功能神经元网络发育中化学诱导变化的细胞和分子机制的了解。小鼠神经前体细胞（mNPC）分化为神经系统特异性细胞类型，并已被证明有价值的检测DNT使用生化和形态学技术。因此，我们研究了一些功能性神经元参数在原代mNPC，以探索其适用性的神经生理学在体外DNT测试。免疫细胞化学证实，mNPC表达神经元，神经胶质细胞和祖细胞标志物在不同的分化持续时间（1，7，14，和21天）。由于细胞内钙（[Ca-2]（i））在神经元发育和功能中起着至关重要的作用，因此我们使用Ca-2响应染料Fura-2在这些分化持续时间内测量了刺激诱发的[Ca-2]（i）变化。去极化、ATP、l-谷氨酸、乙酰胆碱和多巴胺（分别高达87%、57%、93%、28%和37%的反应细胞）引起[Ca-2]（i）的增加（平均范围为65至226 nM），血清素和γ-氨基丁酸（均高达10%的反应细胞）也引起了[Ca-2]（i）的增加。值得注意的是，响应细胞的百分比及其响应幅度随时间的变化表明在体外分化期间相应神经递质受体和相关钙信号传导途径的表达和功能的变化。使用多电极阵列证实了功能性细胞间信号传导途径的发展，表明mNPC在分化12周内产生电活性（在分化14天时55%活性威尔斯孔;平均尖峰速率为1.16尖峰/s/电极）。综合数据表明，mNPC在体外开发功能性神经元特征，使其成为一个有前途的模型，研究化学诱导的神经元功能的发展的影响。
In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous systemspecific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing. Immunocytochemistry confirmed that mNPCs express neuronal, glial, and progenitor markers at various differentiation durations (1, 7, 14, and 21 days). Because intracellular calcium ([Ca-2](i)) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca-2](i) at these differentiation durations using the Ca-2-responsive dye Fura-2. Increases in [Ca-2](i) (averages ranging from 65 to 226nM) were evoked by depolarization, ATP, l-glutamic acid, acetylcholine, and dopamine (up to 87%, 57%, 93%, 28%, and 37% responding cells, respectively) and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multielectrode arrays, demonstrating that mNPCs develop electrical activity within 12 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode). The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the development of neuronal function.