Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43
Immunogold evidence that neuronal gap junctions in adult rat brain and spinal cord contain connexin-36 but not connexin-32 or connexin-43
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DOI：
10.1073/pnas.97.13.7573
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发表时间：
2000-06-20
影响因子：
11.1
通讯作者：
Nagy, JI
中科院分区：
文献类型：
--
作者：
Rash, JE;Staines, WA;Nagy, JI
生理和超微结构证据表明，缝隙连接连接了哺乳动物中枢神经系统(CNS)中的许多类别的神经元，允许直接的电和代谢通讯。在成年大鼠脑内至少六种形成缝隙连接的连接蛋白中，连接蛋白-(Cx)-32、Cx36和Cx43已被报道存在于神经元中。然而，在超微结构定义的神经元缝隙连接处没有连接蛋白的报道。为了直接解决这个问题，我们使用冷冻骨折复制免疫金标记法(FRIL)和免疫荧光法(IF)对Cx36在大鼠脑和脊髓中的亚细胞和区域定位进行了可视化。针对Cx36的不同序列产生了三种抗体。通过Western blotting，这些抗体检测到36 kDa和66 kDa的蛋白质，分别对应于Cx36单体和二聚体。FRIL对Cx36和Cx43进行双重标记后，发现下橄榄的神经元缝隙连接。脊髓和视网膜一直被免疫金标记物Cx36标记，但没有Cx43标记，反之亦然。Cx43在星形胶质细胞和室管膜细胞缝隙连接中表达，而Cx36在星形胶质细胞和室管膜细胞缝隙连接中未表达。在来自10个CNS区域的250个Cx32/Cx43单标和双标复本中，没有标记Cx32或Cx43的神经元缝隙连接，相反，Cx32和Cx43仅限于神经胶质缝隙连接。到了如果。Cx36标记广泛分布于神经束中，包括树突和神经元胞体内。根据Cx36在神经元缝隙连接中的FRIL鉴定，如果Cx36在大鼠脑和脊髓中的成像，含有Cx36的神经元缝隙连接似乎以足够的密度出现在成人中枢神经系统中，提供广泛的电和代谢耦合。
Physiological and ultrastructural evidence indicates that gap junctions link many classes of neurons in mammalian central nervous system (CNS), allowing direct electrical and metabolic communication. Among at least six gap junction-forming connexin proteins in adult rat brain, connexin- (Cx) 32, Cx36, and Cx43 have been reported to occur in neurons. However, no connexin has been documented at ultrastructurally defined neuronal gap junctions. To address this question directly, freeze-fracture replica immunogold labeling (FRIL) and immunofluorescence (IF) were used to visualize the subcellular and regional localization of Cx36 in rat brain and spinal cord. Three antibodies were generated against different sequences in Cx36. By Western blotting, these antibodies detected protein at 36 and 66 kDa, corresponding to Cx36 monomer and dimer forms, respectively. After double-labeling for Cx36 and Cx43 by FRIL, neuronal gap junctions in inferior olive. spinal cord, and retina were consistently immunogold-labeled for Cx36, but none were labeled for Cx43, Conversely. Cx43 but not Cx36 was detected in astrocyte and ependymocyte gap junctions. In >250 Cx32/Cx43 single- and double-labeled replicas from 10 CNS regions, no neuronal gap junctions were labeled for either Cx32 or Cx43, Instead, Cx32 and Cx43 were restricted to glial gap junctions. By IF. Cx36 labeling was widely distributed in neuropil, including along dendritic processes and within neuronal somata, On the basis of FRIL identification of Cx36 in neuronal gap junctions and IF imaging of Cx36 throughout rat brain and spinal cord, neuronal gap junctions containing Cx36 appear to occur in sufficient density to provide widespread electrical and metabolic coupling in adult CNS.