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

使用前脑啡肽原启动子 GFP 载体可视化含脑啡肽的神经元

基本信息

批准号：
12680798
负责人：
HORI Yuuichi
金额：
$ 2.3万
依托单位：
Dokkyo Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

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

最近，生物射弹基因枪已被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。在本研究中，我们用由增强型绿色荧光蛋白（GFP）驱动的增强型绿色荧光蛋白（GFP）基因转染小鼠脊髓切片的培养物。前脑啡肽启动子，使用适用于浅表背角小神经元的颗粒介导的基因转移系统。浅表背角表达高密度的 GFP 荧光，让人想起之前报道的分布相反，用 CMV 启动子 GFP 质粒转染的切片在整个脊髓中具有均匀的 GFP 荧光分布。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 表达受前脑啡肽启动子及其 cAMP 响应元件。表达 GFP 的神经元的胞体直径范围为 5μm 至 12μm … 更多。表达GFP的神经元的细胞体和神经突形态各异，大部分类似于“柄细胞”和“胰岛细胞”，S. Gobel在脊髓浅表背角中描述了这些细胞。单细胞RT-PCR分析表明，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 NR2B 亚基的发生率明显更高。 NMDA受体亚基在脑啡肽能神经元和非脑啡肽能神经元之间受到差异性调节，尽管这种差异调节的功能意义目前尚不清楚。最后，本研究中描述的前脑啡肽原启动子-GFP质粒转染的脊髓的器官切片培养物提供了研究前脑啡肽原基因表达的跨突触调节的机会，并能够快速分析前脑啡肽原基因表达的变化来自中枢神经系统特定区域的高度受限的神经元群体。