Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells
Functional NMDA receptor channels generated by NMDAR2B gene transfer in rat cerebellar Purkinje cells
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大鼠小脑浦肯野细胞中 NMDAR2B 基因转移产生的功能性 NMDA 受体通道
DOI：
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发表时间：
2003
影响因子：
3.4
通讯作者：
S. Ozawa
中科院分区：
文献类型：
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作者：
W. Kakegawa;K. Tsuzuki;M. Iino;S. Ozawa
成年小脑浦肯野细胞是中枢神经系统中的特殊神经元，因为它表达高水平的NMDAR 1（NR 1）mRNA而不表达任何NMDAR 2（NR 2）mRNA。它没有功能性NMDA受体（NMDAR）通道，但它接收大量的兴奋性输入。尽管NR 1 mRNA的高水平表达，NR 1蛋白在成熟浦肯野细胞中的存在和定位是有争议的。为了检测NR 1蛋白的存在及其形成功能性NMDAR的能力，我们通过辛德毕斯病毒介导的基因转移在大鼠成熟浦肯野神经元中表达NR 2B亚基。编码NR 2B和增强型绿色荧光蛋白（GFP）基因的重组病毒（命名为SIN-EG-NR 2B）感染浦肯野细胞而不感染神经胶质细胞。感染后18-24 h，在浦肯野细胞的索马胞体和整个树突中检测到GFP荧光。在大多数GFP阳性细胞中，通过NR 2B特异性抗体的免疫染色检测到NR 2B蛋白的表达。在感染SIN-EG-NR 2B的浦肯野细胞中，NMDA的离子电渗应用诱导了显著的NMDAR介导的电流反应，表明外源性NR 2B与内源性NR 1组装形成功能性NMDAR。此外，在感染的浦肯野细胞中，在攀爬纤维和平行纤维突触中都检测到NMDAR介导的突触电流。因此，成熟的浦肯野细胞产生NR 1蛋白，其准备与NR 2联合收割机在兴奋性突触中形成功能性NMDAR。
The adult cerebellar Purkinje cell is an exceptional neuron in the central nervous system in that it expresses high levels of NMDAR1 (NR1) mRNA without expressing any NMDAR2 (NR2) mRNAs. It has no functional NMDA receptor (NMDAR) channels, although it receives enormous numbers of excitatory inputs. Despite the high level of NR1 mRNA expression, the presence and localization of NR1 protein in mature Purkinje cells are controversial. To examine the presence of NR1 protein and its ability to form functional NMDARs, we expressed the NR2B subunit in rat mature Purkinje neurons by Sindbis viral‐mediated gene transfer. The recombinant virus encoding both the NR2B and enhanced green fluorescent protein (GFP) genes (designated as SIN–EG–NR2B) infected Purkinje cells without infecting glial cells. GFP fluorescence was detected in the soma and throughout dendrites of Purkinje cells 18–24 h postinfection. In most of GFP‐positive cells, the expression of NR2B protein was detected by immunostaining with NR2B‐specific antibodies. In Purkinje cells infected with SIN–EG–NR2B, the iontophoretic application of NMDA induced prominent NMDAR‐mediated current responses, indicating that the exogenous NR2B was assembled with endogenous NR1 to form functional NMDARs. Furthermore, NMDAR‐mediated synaptic currents were detected at both the climbing fibre and parallel fibre synapses in infected Purkinje cells. Thus, the mature Purkinje cell produces NR1 protein that is ready to combine with NR2 to form functional NMDARs in excitatory synapses.
影响因子：
2.5
作者：
Vicini, S;Wang, JF;Grayson, DR
通讯作者：
Grayson, DR