Hassoun Amani T, Erdélyi Ferenc, Szabó Gábor, Davis Margaret I
Laboratory for Integrative Neuroscience/Section on Synaptic Pharmacology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892, USA.
J Neurosci Methods. 2007 Nov 30;166(2):178-94. doi: 10.1016/j.jneumeth.2007.07.008. Epub 2007 Jul 22.
We developed and characterized an assay that allows for rapid examination of migration of specific neuronal populations within a mixed population using the Boyden chamber principle. Migration of cerebellar interneurons and granule cells was examined using mice expressing enhanced green fluorescent protein (eGFP) under the glutamate decarboxylase (GAD(65)) and growth-associated protein-43 (GAP43) promoters, respectively. Brain-derived neurotrophic factor (BDNF) was used as the prototypic motogen for both populations. Fluorescent light-blocking inserts (FluoroBlok) with different pore sizes and densities were compared in a two-compartment assay. Immunodetection of polarity markers and nuclear staining indicated that dendrites and somata are preferentially extended through the pores in response to BDNF. Inserts coated with extracellular matrix (ECM) proteins were used to examine interactions between BDNF and the ECM during migration. ECM proteins alone stimulated migration when the lower side of the insert was coated, however coating of both sides of the insert slowed migration when compared to poly-D-lysine. Addition of a PI 3-kinase inhibitor to the lower compartment blocked BDNF-stimulated migration of both populations while a Src inhibitor reduced laminin-stimulated migration of interneurons, but not granule cells. We also examined use of neurons cultured from GAD(65)-eGFP mice as a reporter system for promoter activity. GAD(65)-eGFP mice may also be useful as a model for promoter regulation and the potential confounding effects of eGFP induction by the stimuli are also addressed. This assay allows for rapid analysis of motogens, substrates and signaling pathways that regulate migration of selected neuronal populations.
我们开发并表征了一种检测方法，该方法利用博伊登小室原理，能够快速检测混合群体中特定神经元群体的迁移情况。分别使用在谷氨酸脱羧酶（GAD(65)）和生长相关蛋白43（GAP43）启动子控制下表达增强型绿色荧光蛋白（eGFP）的小鼠，检测小脑中间神经元和颗粒细胞的迁移。脑源性神经营养因子（BDNF）被用作这两种细胞群体的典型促细胞运动因子。在两室检测中比较了具有不同孔径和密度的荧光光阻断插入物（FluoroBlok）。极性标记物的免疫检测和细胞核染色表明，树突和胞体在BDNF的作用下优先通过孔隙延伸。使用包被有细胞外基质（ECM）蛋白的插入物来检测迁移过程中BDNF与ECM之间的相互作用。当插入物的下侧包被ECM蛋白时，其单独就能刺激细胞迁移，然而与聚-D-赖氨酸相比，插入物两侧都包被时会减缓细胞迁移。向下室添加PI 3-激酶抑制剂可阻断BDNF刺激的两种细胞群体的迁移，而Src抑制剂可减少层粘连蛋白刺激的中间神经元迁移，但对颗粒细胞无此作用。我们还检测了使用从GAD(65)-eGFP小鼠培养的神经元作为启动子活性报告系统的情况。GAD(65)-eGFP小鼠也可能作为启动子调控的模型，并且还探讨了刺激诱导eGFP产生的潜在混杂效应。该检测方法能够快速分析调节选定神经元群体迁移的促细胞运动因子、底物和信号通路。
