NMDA受体在大鼠和鲫鱼视网膜上的表达分布
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摘要
目的：研究离子型谷氨酸受体NMDA (N-methyl-D-aspartate)受体的功能性亚基1(NMDAR1,NR1)蛋白在大鼠视网膜上的表达分布情况。
方法：采用western blot技术对大鼠视网膜组织匀浆行免疫印迹,验证所用抗NR1抗体的特异性。用免疫组化荧光单标技术,在SD大鼠视网膜垂直冰冻切片上研究NR1蛋白在大鼠视网膜上的大致分布,又使用免疫荧光双标技术研究其在视网膜双极细胞(bipolar cell, BC)、无长突细胞(amacrine cell, AC)及神经节细胞(ganglion cell, GC)上的表达分布。
结果：western blot分析结果显示本实验所用NR1抗体有较好的特异性。免疫荧光单标法结果显示NR1亚基的免疫阳性产物弥散分布在大鼠视网膜的外网层(outer plexiform layer, OPL)和内网层(inner plexiform layer, IPL)全层,且内核层(inner nuclear layer, INL)及神经节细胞层(ganglion cell layer, GCL)的不同种类细胞均有NR1蛋白的分布,但外核层(outer nuclear layer, ONL)没有观察到明显的NR1的阳性反应。进一步采用细胞特异性标记物和NR1抗体免疫双标,显示CHX10阳性的双极细胞胞体、PKCa阳性的视杆信号主导和视锥信号主导的ON型双极细胞(rod-dominant bipolar cell, RBC; cone-domidant ON type bipolar cell, ON-CBC)、recoverin阳性的ON-CBC和视锥信号主导的OFF型双极细胞(cone-dominant OFF type bipolar cell, OFF-CBC)和NR1均有共表达。我们观察到胆碱能、多巴胺能、γ-氨基丁酸能和甘氨酸能的无长突细胞均和NR1有共表达。用于标记神经节细胞的特异性标记物Bm3a阳性的细胞也有NR1蛋白的表达。
结论：NMDA受体参与了外层及内层视网膜的信息传递。研究提供了NR1亚基参与ON型和OFF型双极细胞两条通路的证据,有力支持了NMDA受体在视网膜信号传导通路中的调节作用。NR1蛋白在神经节细胞的表达则表明NMDA受体也很可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。
目的：研究离子型谷氨酸受体NMDA受体的调节性亚基3A和3B(NMDAR3A-NMDAR3B, NR3A-NR3B)蛋白在鲫鱼视网膜上的表达分布情况。
方法：采用western blot技术对鲫鱼视网膜组织匀浆行免疫印迹,验证所用抗NR3A和抗NR3B抗体的特异性。用免疫组化荧光单标及共聚焦激光扫描显微技术,在鲫鱼视网膜垂直冰冻切片上研究NR3A和NR3B蛋白在鲫鱼视网膜特别是外层视网膜上的表达分布。
结果：western blot分析结果显示本实验所用抗NR3A和抗NR3B抗体分别较特异性地标记鲫鱼视网膜中的NR3A和NR3B蛋白。免疫荧光法结果显示NR3A的免疫阳性产物弥散分布在鲫鱼视网膜的外网层,呈点状或弥散状分布在内网层,内核层及神经节细胞层的不同种类细胞均有NR3A蛋白的分布。重点可见外核层细胞胞体、轴突和轴突终末上有NR3A的强阳性免疫反应。NR3B的免疫阳性产物弥散性分布在OPL和IPL,亦分布在INL和GCL少许细胞上,但阳性信号均比较弱。重点可见ONL只有部分光感受器细胞的胞体和轴突有较强的免疫阳性反应。
结论：NR3A和NR3B蛋白的广泛表达进一步表明NMDA参与外层及内层视网膜的信息传递。NR3亚基在光感受器细胞的表达表明其可能参与年龄相关性黄斑变性和遗传性视网膜退行性疾病发病过程中兴奋毒性导致的光感受器细胞凋亡,其在神经节细胞层的表达则表明NR3亚基也可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。
Objective:N-methyl-D-aspartate receptors (NMDARs), are one of the ionotropic glutamate receptors, which play key roles in the neuronal communication in the retina. The aim of this study is to investigate NMDA receptor subunit1(NR1) protein expression and distribution in the rat retina.
Methods:We examined the qualitative expression and spatial distribution of NR1protein in the rat retina by western blot analysis and immunofluorescence double labeling respectively.
Results:Western blot analysis declared that NR1protein was present in the rat retina, and the specificity of the NR1antibody. We showed that labeling for NR1was diffusely distributed in the outer plexiform layer (OPL) and throughout full thickness of the inner plexiform layer (IPL). The NR1-immunoreactivity (IR) was also displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR1was expressed in both rod and cone bipolar cells identified by specific bipolar cell markers Chx10, protein kinase C (PKC) and recoverin. All the amacrine cells that we studied, including cholinergic, dopaminergic, GABAergic and glycinergic amacrine cells, were NR1-IR positive. In the ganglion cell layer, NR1-IR was expressed in all cells that were positive for the ganglion cell maker Brn3a.
Conclusion:Our work suggests that the NR1subunit is expressed more widely than was previously appreciated, which providing additional evidence for a role of NR1in neuronal transmitter and synaptic plasticity. We provide important evidence showing that retina bipolar cells could have NMDA receptor expression, that involve in modulation of vertical retina signal transmission.
Objective:The aim of this study is to investigate ionotropic glutamate receptors NMDA receptor regulation subunits3A and3B (NR3A-NR3B) protein expression and distribution.in the carp retina.
Methods:We examined the qualitative expression and spatial distribution of NR3A and NR3B protein in the carp retina by western blot analysis and immunofluorescence labeling respectively.
Results:Western blot analysis declared that NR3A and NR3B proteins were present in the carp retina, and the specificity of these two antibodies. We showed that labeling for NR3A was diffusely distributed in the outer plexiform layer (OPL) and a punctate or diffusion-like distribution in the inner plexiform layer (IPL). The NR3A-immunoreactivity (IR) was also displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR3A was strongly expressed in the cell bodies, axons and axon terminals of the outer nuclear layer (ONL). The NR3B immunoreactive positive products were diffusely distributed in the OPL and IPL, also in a few cells in the INL and GCL, but the positive signals were weak. Some cells of ONL have a stronger immune positive reaction.
Conclusion:Our work suggests that the NR3A and NR3B subunits are widely expressed in the carp retina, which providing additional evidence for a role of NR3in neuronal transmitter and synaptic plasticity. We provide important evidences showing that NR3subunits expression in photoreceptor cells may be involved in the excitotoxicity in the course of age-related macular degeneration and inherited retinal degenerative diseases, and their expression in ganglion cells indicate that NR3subunits is also likely to participate in the ganglion cell apoptosis caused by excitotoxicity in glaucoma or retinal ischemic diseases.
方法：采用western blot技术对大鼠视网膜组织匀浆行免疫印迹,验证所用抗NR1抗体的特异性。用免疫组化荧光单标技术,在SD大鼠视网膜垂直冰冻切片上研究NR1蛋白在大鼠视网膜上的大致分布,又使用免疫荧光双标技术研究其在视网膜双极细胞(bipolar cell, BC)、无长突细胞(amacrine cell, AC)及神经节细胞(ganglion cell, GC)上的表达分布。
结果：western blot分析结果显示本实验所用NR1抗体有较好的特异性。免疫荧光单标法结果显示NR1亚基的免疫阳性产物弥散分布在大鼠视网膜的外网层(outer plexiform layer, OPL)和内网层(inner plexiform layer, IPL)全层,且内核层(inner nuclear layer, INL)及神经节细胞层(ganglion cell layer, GCL)的不同种类细胞均有NR1蛋白的分布,但外核层(outer nuclear layer, ONL)没有观察到明显的NR1的阳性反应。进一步采用细胞特异性标记物和NR1抗体免疫双标,显示CHX10阳性的双极细胞胞体、PKCa阳性的视杆信号主导和视锥信号主导的ON型双极细胞(rod-dominant bipolar cell, RBC; cone-domidant ON type bipolar cell, ON-CBC)、recoverin阳性的ON-CBC和视锥信号主导的OFF型双极细胞(cone-dominant OFF type bipolar cell, OFF-CBC)和NR1均有共表达。我们观察到胆碱能、多巴胺能、γ-氨基丁酸能和甘氨酸能的无长突细胞均和NR1有共表达。用于标记神经节细胞的特异性标记物Bm3a阳性的细胞也有NR1蛋白的表达。
结论：NMDA受体参与了外层及内层视网膜的信息传递。研究提供了NR1亚基参与ON型和OFF型双极细胞两条通路的证据,有力支持了NMDA受体在视网膜信号传导通路中的调节作用。NR1蛋白在神经节细胞的表达则表明NMDA受体也很可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。
目的：研究离子型谷氨酸受体NMDA受体的调节性亚基3A和3B(NMDAR3A-NMDAR3B, NR3A-NR3B)蛋白在鲫鱼视网膜上的表达分布情况。
方法：采用western blot技术对鲫鱼视网膜组织匀浆行免疫印迹,验证所用抗NR3A和抗NR3B抗体的特异性。用免疫组化荧光单标及共聚焦激光扫描显微技术,在鲫鱼视网膜垂直冰冻切片上研究NR3A和NR3B蛋白在鲫鱼视网膜特别是外层视网膜上的表达分布。
结果：western blot分析结果显示本实验所用抗NR3A和抗NR3B抗体分别较特异性地标记鲫鱼视网膜中的NR3A和NR3B蛋白。免疫荧光法结果显示NR3A的免疫阳性产物弥散分布在鲫鱼视网膜的外网层,呈点状或弥散状分布在内网层,内核层及神经节细胞层的不同种类细胞均有NR3A蛋白的分布。重点可见外核层细胞胞体、轴突和轴突终末上有NR3A的强阳性免疫反应。NR3B的免疫阳性产物弥散性分布在OPL和IPL,亦分布在INL和GCL少许细胞上,但阳性信号均比较弱。重点可见ONL只有部分光感受器细胞的胞体和轴突有较强的免疫阳性反应。
结论：NR3A和NR3B蛋白的广泛表达进一步表明NMDA参与外层及内层视网膜的信息传递。NR3亚基在光感受器细胞的表达表明其可能参与年龄相关性黄斑变性和遗传性视网膜退行性疾病发病过程中兴奋毒性导致的光感受器细胞凋亡,其在神经节细胞层的表达则表明NR3亚基也可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。
Objective:N-methyl-D-aspartate receptors (NMDARs), are one of the ionotropic glutamate receptors, which play key roles in the neuronal communication in the retina. The aim of this study is to investigate NMDA receptor subunit1(NR1) protein expression and distribution in the rat retina.
Methods:We examined the qualitative expression and spatial distribution of NR1protein in the rat retina by western blot analysis and immunofluorescence double labeling respectively.
Results:Western blot analysis declared that NR1protein was present in the rat retina, and the specificity of the NR1antibody. We showed that labeling for NR1was diffusely distributed in the outer plexiform layer (OPL) and throughout full thickness of the inner plexiform layer (IPL). The NR1-immunoreactivity (IR) was also displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR1was expressed in both rod and cone bipolar cells identified by specific bipolar cell markers Chx10, protein kinase C (PKC) and recoverin. All the amacrine cells that we studied, including cholinergic, dopaminergic, GABAergic and glycinergic amacrine cells, were NR1-IR positive. In the ganglion cell layer, NR1-IR was expressed in all cells that were positive for the ganglion cell maker Brn3a.
Conclusion:Our work suggests that the NR1subunit is expressed more widely than was previously appreciated, which providing additional evidence for a role of NR1in neuronal transmitter and synaptic plasticity. We provide important evidence showing that retina bipolar cells could have NMDA receptor expression, that involve in modulation of vertical retina signal transmission.
Objective:The aim of this study is to investigate ionotropic glutamate receptors NMDA receptor regulation subunits3A and3B (NR3A-NR3B) protein expression and distribution.in the carp retina.
Methods:We examined the qualitative expression and spatial distribution of NR3A and NR3B protein in the carp retina by western blot analysis and immunofluorescence labeling respectively.
Results:Western blot analysis declared that NR3A and NR3B proteins were present in the carp retina, and the specificity of these two antibodies. We showed that labeling for NR3A was diffusely distributed in the outer plexiform layer (OPL) and a punctate or diffusion-like distribution in the inner plexiform layer (IPL). The NR3A-immunoreactivity (IR) was also displayed in a variety of cells in the inner nuclear layer (INL) and the ganglion cell layer (GCL). Interestingly, NR3A was strongly expressed in the cell bodies, axons and axon terminals of the outer nuclear layer (ONL). The NR3B immunoreactive positive products were diffusely distributed in the OPL and IPL, also in a few cells in the INL and GCL, but the positive signals were weak. Some cells of ONL have a stronger immune positive reaction.
Conclusion:Our work suggests that the NR3A and NR3B subunits are widely expressed in the carp retina, which providing additional evidence for a role of NR3in neuronal transmitter and synaptic plasticity. We provide important evidences showing that NR3subunits expression in photoreceptor cells may be involved in the excitotoxicity in the course of age-related macular degeneration and inherited retinal degenerative diseases, and their expression in ganglion cells indicate that NR3subunits is also likely to participate in the ganglion cell apoptosis caused by excitotoxicity in glaucoma or retinal ischemic diseases.
引文
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