Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures
Automated Live-Cell Imaging of Synapses in Rat and Human Neuronal Cultures
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DOI：
10.3389/fncel.2019.00467
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发表时间：
2019-10-17
影响因子：
5.3
通讯作者：
Thayer, Stanley A.
中科院分区：
文献类型：
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作者：
Green, Matthew V.;Pengo, Thomas;Thayer, Stanley A.
突触丢失和树突损伤与许多神经退行性疾病中的认知下降相关，是神经发育障碍的基础，并与环境和药物诱导的CNS毒性相关。然而，缺乏设计用于测量活细胞之间突触连接损失的筛选测定。在这里，我们描述了设计和验证的自动突触成像分析（ASIA），一种有效的方法来标记，图像和分析活神经元之间的突触。使用病毒转导表达标记突触的荧光蛋白和自动计算机控制的显微镜，我们开发了一种方法来识别调节突触数量的试剂。ASIA与共焦和宽视场显微镜兼容;宽视场图像采集更快，但需要在分析中进行解卷积步骤。这两种类型的图像都输入到可以在ImageJ、CellProfiler和MetaMorph平台上运行的批处理分析软件中。主要分析终点是结构突触的数量和细胞活力。因此，明显的细胞死亡与突触密度的细微变化不同，这是研究神经退行性过程时的一个重要区别。在用100 mM 2-溴棕榈酸（2-BP）（一种防止突触后密度95（PSD 95）聚集的化合物）处理24小时的大鼠海马培养物中，ASIA可靠地检测到在不存在细胞死亡的情况下突触后密度95增强的绿色荧光蛋白（PSD 95-eGFP）标记的突触的丢失。相比之下，用100 mM谷氨酸处理产生突触损失和显著的细胞死亡，这是从共表达mCherry产生的二进制图像中的形态学变化确定的。用3 mM锂处理24 h显著增加荧光斑点的数量，表明ASIA也检测突触发生。概念验证研究表明，细胞特异性启动子使抑制性或主要神经元的选择性研究成为可能，并且替代报告构建体使GABA能或神经元能突触的定量成为可能。ASIA还可用于研究人类诱导多能干细胞（iPSC）衍生的皮质神经元之间的突触丢失。在用100 mM 2-BP或100 mM谷氨酸处理24 h的iPSC衍生的神经元培养物中检测到在不存在细胞死亡的情况下的显著突触损失，而300 mM谷氨酸产生突触损失和细胞死亡。ASIA显示了识别引起突触毒性的药物和筛选预防或逆转突触丧失的化合物的前景。
Synapse loss and dendritic damage correlate with cognitive decline in many neurodegenerative diseases, underlie neurodevelopmental disorders, and are associated with environmental and drug-induced CNS toxicities. However, screening assays designed to measure loss of synaptic connections between live cells are lacking. Here, we describe the design and validation of automated synaptic imaging assay (ASIA), an efficient approach to label, image, and analyze synapses between live neurons. Using viral transduction to express fluorescent proteins that label synapses and an automated computer-controlled microscope, we developed a method to identify agents that regulate synapse number. ASIA is compatible with both confocal and wide-field microscopy; wide-field image acquisition is faster but requires a deconvolution step in the analysis. Both types of images feed into batch processing analysis software that can be run on ImageJ, CellProfiler, and MetaMorph platforms. Primary analysis endpoints are the number of structural synapses and cell viability. Thus, overt cell death is differentiated from subtle changes in synapse density, an important distinction when studying neurodegenerative processes. In rat hippocampal cultures treated for 24 h with 100 mM 2-bromopalmitic acid (2-BP), a compound that prevents clustering of postsynaptic density 95 (PSD95), ASIA reliably detected loss of postsynaptic density 95-enhanced green fluorescent protein (PSD95-eGFP)-labeled synapses in the absence of cell death. In contrast, treatment with 100 mM glutamate produced synapse loss and significant cell death, determined from morphological changes in a binary image created from co-expressed mCherry. Treatment with 3 mM lithium for 24 h significantly increased the number of fluorescent puncta, showing that ASIA also detects synaptogenesis. Proof of concept studies show that cell-specific promoters enable the selective study of inhibitory or principal neurons and that alternative reporter constructs enable quantification of GABAergic or glutamatergic synapses. ASIA can also be used to study synapse loss between human induced pluripotent stem cell (iPSC)-derived cortical neurons. Significant synapse loss in the absence of cell death was detected in the iPSC-derived neuronal cultures treated with either 100 mM 2-BP or 100 mM glutamate for 24 h, while 300 mM glutamate produced synapse loss and cell death. ASIA shows promise for identifying agents that evoke synaptic toxicities and screening for compounds that prevent or reverse synapse loss.