Roqué Pamela J, Guizzetti Marina, Costa Lucio G
Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington.
Curr Protoc Toxicol. 2014 May 27;60:12.22.1-32. doi: 10.1002/0471140856.tx1222s60.
Behavioral problems (e.g., learning and memory) following developmental exposure to toxicants suggests that dysregulation of the process of synapse formation and function may occur. The ability to assess these changes is thus of value. This unit describes a method to investigate toxicant-induced changes to synaptic structure formation in primary hippocampal neurons using immunocytochemical labeling of the pre- and post-synaptic markers synaptophysin and PSD-95, confocal imaging, and three-dimensional object analysis. Protocols for the long-term culturing of primary hippocampal neurons and of primary cortical astrocytes, as well as their co-culture, are included. While the described methods focus on how astrocytes influence synapse formation and how toxicants may interfere in this process, modifications to the experimental plan can easily be implemented. This would allow for the investigation of the effects of toxicants after treating neurons alone, or both astrocytes and neurons in co-culture. With the common endpoint of synapse structure formation, differences between varying treatment paradigms can expand the understanding of the influence of particular toxicants on these diverse cell types and provide insight into potential mechanisms of effect and the contributions of each to synapse formation.
发育过程中接触毒物后出现的行为问题（如学习和记忆方面）表明，突触形成和功能过程可能发生失调。因此，评估这些变化的能力具有重要价值。本单元介绍了一种方法，通过对突触前和突触后标记物突触素和突触后密度蛋白95进行免疫细胞化学标记、共聚焦成像和三维物体分析，来研究毒物对原代海马神经元突触结构形成的影响。其中还包括原代海马神经元和原代皮质星形胶质细胞的长期培养方案以及它们的共培养方案。虽然所描述的方法侧重于星形胶质细胞如何影响突触形成以及毒物如何干扰这一过程，但可以很容易地对实验方案进行修改。这将允许在单独处理神经元或共培养的星形胶质细胞和神经元后研究毒物的作用。以突触结构形成为共同终点，不同处理模式之间的差异可以扩展对特定毒物对这些不同细胞类型影响的理解，并深入了解潜在的作用机制以及每种细胞对突触形成的贡献。
