In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.
In vitro assessment of developmental neurotoxicity: use of microelectrode arrays to measure functional changes in neuronal network ontogeny.
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DOI：
10.3389/fneng.2011.00001
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Shafer TJ
中科院分区：
文献类型：
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作者：
Robinette BL;Harrill JA;Mundy WR;Shafer TJ
由于发育性神经毒性测试指南需要大量的动物实验，而且费用昂贵，因此开发体外方法来筛选潜在的发育性神经毒性化学物质是一个高度优先考虑的问题。许多建议的筛选方法是生化或形态学的，并没有评估神经网络的功能。在这项研究中，微电极阵列（MEAs）被用来确定是否可以通过评估自发网络活动的发展来检测化学诱导的功能变化。mea记录神经元网络中的单个动作电位峰值以及组动作电位峰值（爆发），并且可以在体外（DIV）中重复评估活动。在MEAs上制备大鼠皮层神经元原代培养物，并在DIV 2、6、9、13和20上评估自发活性，以确定皮层网络自发峰和破裂的体外发育特征。此外，将5 μM的蛋白激酶C抑制剂bisindolylmaleamide-1 （Bis-1）加入到DIV 5上的MEAs （n = 9-18）中，以确定是否可以检测到自发活性的变化，以响应神经突生长的抑制。在对照MEAs中，活性通道的数量从DIV 2的0/MEA增加到DIV 13的37±5/MEA；在DIV 6和9之间增加速度最快，而在DIV 20之间活性下降。爆发通道的数量以及爆发的总数也观察到类似的模式。Bis-1减少了有效信道/MEA和爆发信道/MEA的数量。突发特性，如突发持续时间和突发尖峰的数量，被Bis-1改变。这些结果表明，MEAs可以用来评估体外功能神经元网络的发育，以及化学诱导的功能障碍。
Because the Developmental Neurotoxicity Testing Guidelines require large numbers of animals and is expensive, development of in vitro approaches to screen chemicals for potential developmental neurotoxicity is a high priority. Many proposed approaches for screening are biochemical or morphological, and do not assess function of neuronal networks. In this study, microelectrode arrays (MEAs) were used to determine if chemical-induced changes in function could be detected by assessing the development of spontaneous network activity. MEAs record individual action potential spikes as well as groups of spikes (bursts) in neuronal networks, and activity can be assessed repeatedly over days in vitro (DIV). Primary cultures of rat cortical neurons were prepared on MEAs and spontaneous activity was assessed on DIV 2, 6, 9, 13, and 20 to determine the in vitro developmental profile of spontaneous spiking and bursting in cortical networks. In addition, 5 μM of the protein kinase C inhibitor bisindolylmaleamide-1 (Bis-1) was added to MEAs (n = 9–18) on DIV 5 to determine if changes in spontaneous activity could be detected in response to inhibition of neurite outgrowth. A clear profile of in vitro activity development occurred in control MEAs, with the number of active channels increasing from 0/MEA on DIV 2 to 37 ± 5/MEA by DIV 13; the rate of increase was most rapid between DIV 6 and 9, and activity declined by DIV 20. A similar pattern was observed for the number of bursting channels, as well as the total number of bursts. Bis-1 decreased the number of active channels/MEA and the number of bursting channels/MEA. Burst characteristics, such as burst duration and the number of spikes in a burst, were unchanged by Bis-1. These results demonstrate that MEAs can be used to assess the development of functional neuronal networks in vitro, as well as chemical-induced dysfunction.