Visualization of enkephalin-containing neurons using preproenkephalin-promoter GFP vector

最近，生物基因枪已用于转染器官型培养物中中枢神经系统各个区域的神经元。本研究采用颗粒介导的基因转移系统，将增强型绿色荧光蛋白（GFP）基因转染小鼠脊髓片，在背角浅层小神经元中表达高密度的GFP荧光，与以往报道的脑啡肽能神经元的分布相似。而转染CMV启动子GFP质粒的脊髓切片中GFP荧光分布均匀，表达GFP的神经元数量随着腺苷酸环化酶的激活而增加，表明GFP的表达受前脑啡肽原启动子及其cAMP反应元件的控制，GFP表达神经元的索马体直径为5μm ~ 12μm ...更多信息 .虽然GFP表达神经元的胞体和突起形态各异，但它们大多类似于S.单细胞RT-PCR分析显示，与不表达脑啡肽的神经元相比，表达脑啡肽的神经元中N-甲基-D-天冬氨酸（NMDA）受体NR 2B亚基的发生率显著更大。很明显，NMDA受体亚单位的表达在脑啡肽能神经元和非脑啡肽能神经元之间受到差异调节，尽管这种差异调节的功能意义目前尚不清楚。本研究中描述的用前脑啡肽原启动子-GFP质粒转染的脊髓器官型切片培养提供了一个研究转基因的机会。突触调节前脑啡肽原基因表达，并使快速分析的变化前脑啡肽原基因表达的高度限制群体的神经元从特定区域的中枢神经系统。少

Recently, the biolistic gene gun has been used to transfect neurons from various regions of the central nervous system in organotypic cultures. In the present study, we transfected cultures of mouse spinal cord slices with the enhanced green fluorescent protein (GFP) gene driven by the promoter for preproenkephalin, using the particle-mediated gene transfer system adapted for small neurons in the superficial dorsal horn.A high density of GFP fluorescence was expressed in the superficial dorsal horn, reminiscent of the previously reported distribution of enkephalinergic neurons. In contrast, the slices transfected with CMV promoter GFP plasmid had a uniform distribution of GFP fluorescence throughout the spinal cord.The number of GFP-expressing neurons increased in response to activation of adenylate cyclase, suggesting that GFP expression was controlled by the preproenkephalin promoter with its cAMP responsive elements.The soma diameter of GFP-expressing neurons ranged from 5μm to 12μm … More . While GFP-expressing neurons varied in the morphology of cell body and neurite, most of them resembled "stalked cells" and "islet cells", which have been described in the spinal cord superficial dorsal horn by S. Gobel.Single-cell RT-PCR analysis showed that the incidence of N-methyl-D-aspartate (NMDA) receptor NR2B subunit was significantly larger in enkephalin-expressing neurons compared with neurons not expressing enkephalin. It was apparent that expression of the NMDA receptor subunit is differentially regulated between enkephalinergic neurons and non-enkephalinergic neurons, although the functional significance of this differential regulation is unknown at present.Finally, the organotypic slice culture of spinal cord transfected with preproenkephalin-promoter-GFP plasmid described in the present study provided an opportunity to investigate trans-synaptical regulation of preproenkepahlin gene expression and enabled rapid analysis of changes in preproenkephalin gene expression in highly restricted populations of neurons from specific areas of the CNS. Less