TY - JOUR AB - Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM. AU - Tabata, Shigekazu AU - Jevtic, Marijo AU - Kurashige, Nobutaka AU - Fuchida, Hirokazu AU - Kido, Munetsugu AU - Tani, Kazushi AU - Zenmyo, Naoki AU - Uchinomiya, Shohei AU - Harada, Harumi AU - Itakura, Makoto AU - Hamachi, Itaru AU - Shigemoto, Ryuichi AU - Ojida, Akio ID - 7391 IS - 12 JF - iScience SN - 2589-0042 TI - Electron microscopic detection of single membrane proteins by a specific chemical labeling VL - 22 ER - TY - CHAP AB - Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals. AU - Dimitrov, Dimitar AU - Guillaud, Laurent AU - Eguchi, Kohgaku AU - Takahashi, Tomoyuki ED - Skaper, Stephen D. ID - 562 T2 - Neurotrophic Factors TI - Culture of mouse giant central nervous system synapses and application for imaging and electrophysiological analyses VL - 1727 ER - TY - JOUR AB - The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs. AU - Luján, Rafæl AU - Aguado, Carolina AU - Ciruela, Francisco AU - Arus, Xavier AU - Martín Belmonte, Alejandro AU - Alfaro Ruiz, Rocío AU - Martinez Gomez, Jesus AU - De La Ossa, Luis AU - Watanabe, Masahiko AU - Adelman, John AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo ID - 41 JF - Frontiers in Cellular Neuroscience SN - 16625102 TI - Sk2 channels associate with mGlu1α receptors and CaV2.1 channels in Purkinje cells VL - 12 ER - TY - JOUR AB - Three-dimensional (3D) super-resolution microscopy technique structured illumination microscopy (SIM) imaging of dendritic spines along the dendrite has not been previously performed in fixed tissues, mainly due to deterioration of the stripe pattern of the excitation laser induced by light scattering and optical aberrations. To address this issue and solve these optical problems, we applied a novel clearing reagent, LUCID, to fixed brains. In SIM imaging, the penetration depth and the spatial resolution were improved in LUCID-treated slices, and 160-nm spatial resolution was obtained in a large portion of the imaging volume on a single apical dendrite. Furthermore, in a morphological analysis of spine heads of layer V pyramidal neurons (L5PNs) in the medial prefrontal cortex (mPFC) of chronic dexamethasone (Dex)-treated mice, SIM imaging revealed an altered distribution of spine forms that could not be detected by high-NA confocal imaging. Thus, super-resolution SIM imaging represents a promising high-throughput method for revealing spine morphologies in single dendrites. AU - Sawada, Kazuaki AU - Kawakami, Ryosuke AU - Shigemoto, Ryuichi AU - Nemoto, Tomomi ID - 326 IS - 9 JF - European Journal of Neuroscience TI - Super resolution structural analysis of dendritic spines using three-dimensional structured illumination microscopy in cleared mouse brain slices VL - 47 ER - TY - JOUR AB - Although dopamine receptors D1 and D2 play key roles in hippocampal function, their synaptic localization within the hippocampus has not been fully elucidated. In order to understand precise functions of pre- or postsynaptic dopamine receptors (DRs), the development of protocols to differentiate pre- and postsynaptic DRs is essential. So far, most studies on determination and quantification of DRs did not discriminate between subsynaptic localization. Therefore, the aim of the study was to generate a robust workflow for the localization of DRs. This work provides the basis for future work on hippocampal DRs, in light that DRs may have different functions at pre- or postsynaptic sites. Synaptosomes from rat hippocampi isolated by a sucrose gradient protocol were prepared for super-resolution direct stochastic optical reconstruction microscopy (dSTORM) using Bassoon as a presynaptic zone and Homer1 as postsynaptic density marker. Direct labeling of primary validated antibodies against dopamine receptors D1 (D1R) and D2 (D2R) with Alexa Fluor 594 enabled unequivocal assignment of D1R and D2R to both, pre- and postsynaptic sites. D1R immunoreactivity clusters were observed within the presynaptic active zone as well as at perisynaptic sites at the edge of the presynaptic active zone. The results may be useful for the interpretation of previous studies and the design of future work on DRs in the hippocampus. Moreover, the reduction of the complexity of brain tissue by the use of synaptosomal preparations and dSTORM technology may represent a useful tool for synaptic localization of brain proteins. AU - Miklosi, Andras AU - Del Favero, Giorgia AU - Bulat, Tanja AU - Höger, Harald AU - Shigemoto, Ryuichi AU - Marko, Doris AU - Lubec, Gert ID - 705 IS - 6 JF - Molecular Neurobiology TI - Super resolution microscopical localization of dopamine receptors 1 and 2 in rat hippocampal synaptosomes VL - 55 ER -