High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons
High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons
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DOI：
10.1016/j.vascn.2019.106607
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发表时间：
2019-09-01
影响因子：
1.9
通讯作者：
Shirao, Tomoaki
中科院分区：
文献类型：
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作者：
Hanamura, Kenji;Koganezawa, Noriko;Shirao, Tomoaki
前言：检测药物对神经元突触的作用对预测其不良反应具有重要意义。我们用drebrin作为标记物来检测培养神经元突触的变化。高浓度的谷氨酸会减少突触中drebrin的数量。为了提高这种高通量分析方法的可用性，我们将基于drebriin的突触评估应用于微孔板的高含量成像分析。方法：将培养3周的神经元固定，免疫细胞化学染色，可见drebrin簇、树突和神经元细胞体。自动图像采集后，通过定制设计的协议自动测量每个字段的drebrin簇总数，drebrin簇沿树突的线性密度，树突长度和神经元数量。结果：自动图像采集和分析表明，树突长度和沿树突的drebrin簇密度测量一致且可重复。此外，施用10-100 μ M谷氨酸10分钟或0.5-50 μ M拉runculin A 5分钟可显著降低drebrin簇密度，但不影响神经元数量。这些结果与我们之前使用常规荧光显微镜和图像分析软件进行人工图像采集和分析的结果一致。此外，0.3或1.0 μ M staurosporine作用24 h显著减少神经元数量。讨论：本研究表明，利用沿树突的drebrin簇密度高通量成像分析检测物质对突触的影响，足以检测谷氨酸受体激活和latrunculin A治疗的影响，并表明该分析将有助于安全性药理学研究。
Introduction: Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the drebrin-based evaluation of synapses to high-content imaging analysis using microplates.Methods: Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of drebrin clusters per fields, linear density of drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol.Results: Automated image acquisition and analysis showed that dendrite length and drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10-100 mu M glutamate for 10 min or 0.5-50 mu M latrunculin A for 5 min significantly decreased drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 mu M staurosporine for 24 h significantly decreased neuron number.Discussion: The present study demonstrates that this high-throughput imaging analysis of drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.