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，生物学基因枪已被用来转化有机培养中中枢神经系统各个区域的神经元。在本研究中，我们使用颗粒介导的基因转移系统适用于浅表背侧角的小神经元，用preadroenephalin的启动子驱动的绿色荧光蛋白（GFP）基因的增强的小鼠脊髓切片培养。相反，用CMV启动子GFP质粒翻译的切片在整个脊髓中的GFP荧光分布均匀分布。表达GFP的神经元的数量增加了腺苷酸酸环化酶的激活，这表明GFP表达的GFP表达受到了provestemons with somphalin withs procker-diamons diamerts的控制。 5μm至12μm…更多。虽然表达GFP的神经元在细胞体和神经蛋白的形态上有所不同，但大多数类似于“缠扰细胞”和“胰岛细胞”，在脊髓表面背侧角中已经描述了这些细胞。Single-cell-cell rt-PCR分析表明，N-methyl-d---------蛋白NMDA的成员较大，nmdapartate较大的入射与未表达nkephalin的神经元相比，表达kephalin的神经元。 It was apparent that expression of the NMDA receiver subunit is differentially regulated between enkephalin-rgic neurons and non-enkephalin-rgic neurons, although the functional significance of this differential regulation is unknown at present.Finally, the organic slice culture of spinal cord transfected with preproenkephalin-promoter-GFP plasmid described in the present Study provided an opportunity to investigate trans-synaptical调节前晶基因表达的调节，并能够快速分析来自中枢神经系统特定区域的高度限制神经元种群中脑脑基因表达的变化。较少的