Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Budapest, Hungary.
János Szentágothai Doctoral School, Semmelweis University, Budapest, Hungary.
Methods Mol Biol. 2022;2510:341-353. doi: 10.1007/978-1-0716-2384-8_19.
P2X7 receptors regulate different aspects of neuronal development, including neurogenesis, dendritic outgrowth, and axonal elongation. Primary neuronal culture is a widely used model system in neuroscience as it enables to study molecular and cellular events caused by the activation of different ion channels, receptors, and transporters under controlled conditions. Primary neuronal cultures derived from normal and genetically modified mouse models can be used with a wide array of molecular biological, anatomical, and functional techniques such as RNA sequencing, western blots, immunostaining, Ca imaging, and electrophysiology. In addition, they can also be genetically manipulated relatively easily. Moreover, cells can survive for multiple weeks if they are properly maintained and thus the development and maturation of individual neurons and their morphological properties can be studied under different conditions. Here, we present a protocol for the isolation and culturing of primary hippocampal cells from embryonic mouse hippocampal tissue (embryonic days 17.5-18.5). The neurons are plated in poly-L-lysine/laminin coated coverslips, where astroglia proliferation is controlled for the proper study of individual primary neurons. To investigate the development of dendrites and axons, as a good correlate of neuron morphology, we present a transfection protocol, which allows us to fill the whole neuron with a fluorescent protein. Subsequently, we perform tracing and analysis of dendritic branching by Sholl analysis using Neurolucida tracing Software (MBF Bioscience).
P2X7 受体调节神经元发育的不同方面，包括神经发生、树突生长和轴突伸长。原代神经元培养是神经科学中广泛使用的模型系统，因为它可以在受控条件下研究不同离子通道、受体和转运体激活引起的分子和细胞事件。源自正常和基因修饰的小鼠模型的原代神经元培养物可以与广泛的分子生物学、解剖学和功能技术结合使用，例如 RNA 测序、western blot、免疫染色、Ca 成像和电生理学。此外，它们也可以相对容易地进行基因操作。此外，如果细胞得到适当的维持，它们可以存活数周，因此可以在不同条件下研究单个神经元的发育和成熟及其形态特征。在这里，我们提供了从胚胎期 17.5-18.5 天的小鼠海马组织中分离和培养原代海马神经元的方案。神经元被种植在聚-L-赖氨酸/层粘连蛋白包被的盖玻片上，在那里控制星形胶质细胞的增殖，以正确研究单个原代神经元。为了研究树突和轴突的发育，作为神经元形态的良好相关物，我们提出了一种转染方案，该方案允许我们用荧光蛋白填充整个神经元。随后，我们使用 Neurolucida 追踪软件 (MBF Bioscience) 通过 Sholl 分析对树突分支进行追踪和分析。
