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

最近，生物基因枪已被用来在器官型培养中导入中枢神经系统不同区域的神经元。在本研究中，我们利用粒子介导的基因转移系统，将前脑啡肽原启动子驱动的增强型绿色荧光蛋白(GFP)基因导入小鼠脊髓切片培养物中，在背角浅层的小神经元中表达高密度的GFP荧光，这与以前报道的脑啡肽能神经元的分布相似。与之相反，携带CMV启动子gfp基因的脑片上绿色荧光蛋白在脊髓中分布均匀，腺苷环化酶激活后表达绿色荧光蛋白的神经元数量增加，提示绿色荧光蛋白的表达受前脑啡肽启动子及其cAMP反应元件的调控。表达绿色荧光蛋白的神经元胞体直径在5~12μm…之间更多。尽管表达GFP的神经元胞体和轴突的形态各不相同，但它们大多类似于S.Gobel在脊髓浅背角所描述的“柄细胞”和“胰岛细胞”。单细胞RT-PCR分析表明，脑啡肽表达的神经元中N-甲基-D-天冬氨酸(NMDA)受体NR2B亚基的出现率显著高于不表达脑啡肽的神经元。NMDA受体亚单位在脑源性神经元和非脑源性神经元中的表达明显不同，尽管目前这种差异调控的功能意义尚不清楚。最后，本研究描述的转前脑啡肽原启动子-GFP质粒的脊髓器官型切片培养为研究前脑啡肽原基因表达的跨突触调控提供了机会，并使快速分析中枢特定区域高度受限的神经元群体中前脑啡肽原基因表达的变化成为可能。较少

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