PDF(Pubmed)
Abstract：
Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution.
摘要：
小电导钙激活钾（SK）通道对学习和记忆至关重要。然而,它们在神经元中的空间组织的许多方面仍然未知。在这项研究中,我们采取了一种新颖的方法来回答这些问题，将预嵌入免疫金标记与自动双束电子显微镜相结合，该双束电子显微镜集成了聚焦离子束铣削和扫描电子显微镜（FIB/SEM），以同时收集3D图超微结构和生物分子信息。使用这种新方法，我们评估了小鼠CA1区域放射层中存在的6个兴奋性神经元树突状部分和6个抑制性神经元的3D重建中突触外SK2通道的数量和密度的变异性.在整个海马神经元表面观察到SK2免疫颗粒，分散或集群，以及在细胞内位点。定量体积评估显示，棘的突触外SK2通道密度比树突轴高7倍，比中间神经元高35倍。脊柱显示SK2表达的异质群体，一些具有高SK2含量的刺，其他人的内容较低，其他人缺乏SK2渠道。SK2免疫阴性棘明显小于免疫阳性棘。这些结果表明，兴奋性和抑制性神经元之间的SK2通道密度不同，并且表明脊柱中SK2通道的密度存在很大差异。此外，我们证明SK2表达与兴奋性突触有关，但在CA1锥体细胞中没有抑制性突触。因此,SK2通道对兴奋性和突触可塑性的调节预计是神经元类特异性和靶特异性的。这些数据表明免疫金FIB/SEM代表了一种新的强大的EM工具，可以将离子通道的结构和功能与纳米级分辨率相关联。
