Departments of Neurology, New York University Grossman School of Medicine, New York, NY, USA.
Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, United States.
Methods Mol Biol. 2024;2831:21-37. doi: 10.1007/978-1-0716-3969-6_3.
Primary neuronal cultures are commonly used to study genetic and exogenous factors influencing neuronal development and maturation. During development, neurons undergo robust morphological changes involving expansion of dendritic arbor, formation of dendritic spines, and expression of synaptic proteins. In this chapter, we will cover methodological approaches allowing quantitative assessment of in vitro cultured neurons. Various quantitative characteristics of dendritic arbor can be derived based on immunostaining against anti-microtubule-associated protein 2 followed by dendrite tracing with the SNT plug-in of the FIJI software package. The number and subtypes of dendritic spines can be assessed by double labeling with DiI and Phalloidin iFluor448 followed by laser scanning confocal microscopy analysis. Finally, expression of presynaptic and postsynaptic proteins can be determined by immunohistochemistry and quantification using several available software packages including FIJI and Imaris, which also allows for 3D rendering and statistical displaying of the expression level of synaptic proteins.
原代神经元培养常用于研究影响神经元发育和成熟的遗传和外源性因素。在发育过程中，神经元经历了强大的形态变化，包括树突分支的扩展、树突棘的形成和突触蛋白的表达。在本章中，我们将介绍允许定量评估体外培养神经元的方法学方法。基于针对微管相关蛋白 2 的免疫染色，然后使用 FIJI 软件包的 SNT 插件进行树突追踪，可以得出树突分支的各种定量特征。通过 DiI 和 Phalloidin iFluor448 的双重标记，然后进行激光扫描共聚焦显微镜分析，可以评估树突棘的数量和亚型。最后，通过免疫组织化学和几种可用软件包（包括 FIJI 和 Imaris）的定量分析，可以确定突触前和突触后蛋白的表达。这些软件包还允许对突触蛋白的表达水平进行 3D 渲染和统计显示。
