@article{13202,
  abstract     = {Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.},
  author       = {Eguchi, Kohgaku and Le Monnier, Elodie and Shigemoto, Ryuichi},
  issn         = {1529-2401},
  journal      = {The Journal of Neuroscience},
  number       = {23},
  pages        = {4197--4216},
  publisher    = {Society for Neuroscience},
  title        = {{Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons}},
  doi          = {10.1523/JNEUROSCI.1514-22.2023},
  volume       = {43},
  year         = {2023},
}

@article{10889,
  abstract     = {Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.},
  author       = {Shigemoto, Ryuichi},
  issn         = {2050-5701},
  journal      = {Microscopy},
  number       = {Supplement_1},
  pages        = {i72--i80},
  publisher    = {Oxford Academic},
  title        = {{Electron microscopic visualization of single molecules by tag-mediated metal particle labeling}},
  doi          = {10.1093/jmicro/dfab048},
  volume       = {71},
  year         = {2022},
}

@article{10890,
  abstract     = {Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.},
  author       = {Eguchi, Kohgaku and Montanaro-Punzengruber, Jacqueline-Claire and Le Monnier, Elodie and Shigemoto, Ryuichi},
  issn         = {16625129},
  journal      = {Frontiers in Neuroanatomy},
  publisher    = {Frontiers},
  title        = {{The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals}},
  doi          = {10.3389/fnana.2022.846615},
  volume       = {16},
  year         = {2022},
}

@article{7551,
  abstract     = {Novelty facilitates formation of memories. The detection of novelty and storage of contextual memories are both mediated by the hippocampus, yet the mechanisms that link these two functions remain to be defined. Dentate granule cells (GCs) of the dorsal hippocampus fire upon novelty exposure forming engrams of contextual memory. However, their key excitatory inputs from the entorhinal cortex are not responsive to novelty and are insufficient to make dorsal GCs fire reliably. Here we uncover a powerful glutamatergic pathway to dorsal GCs from ventral hippocampal mossy cells (MCs) that relays novelty, and is necessary and sufficient for driving dorsal GCs activation. Furthermore, manipulation of ventral MCs activity bidirectionally regulates novelty-induced contextual memory acquisition. Our results show that ventral MCs activity controls memory formation through an intra-hippocampal interaction mechanism gated by novelty.},
  author       = {Fredes Tolorza, Felipe A and Silva Sifuentes, Maria A and Koppensteiner, Peter and Kobayashi, Kenta and Jösch, Maximilian A and Shigemoto, Ryuichi},
  journal      = {Current Biology},
  number       = {1},
  pages        = {P25--38.E5},
  publisher    = {Elsevier},
  title        = {{Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation}},
  doi          = {10.1016/j.cub.2020.09.074},
  volume       = {31},
  year         = {2021},
}

@article{9330,
  abstract     = {In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.},
  author       = {Schöpf, Clemens L. and Ablinger, Cornelia and Geisler, Stefanie M. and Stanika, Ruslan I. and Campiglio, Marta and Kaufmann, Walter and Nimmervoll, Benedikt and Schlick, Bettina and Brockhaus, Johannes and Missler, Markus and Shigemoto, Ryuichi and Obermair, Gerald J.},
  issn         = {1091-6490},
  journal      = {PNAS},
  number       = {14},
  publisher    = {National Academy of Sciences},
  title        = {{Presynaptic α2δ subunits are key organizers of glutamatergic synapses}},
  doi          = {10.1073/pnas.1920827118},
  volume       = {118},
  year         = {2021},
}

@article{9437,
  abstract     = {The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.},
  author       = {Bhandari, Pradeep and Vandael, David H and Fernández-Fernández, Diego and Fritzius, Thorsten and Kleindienst, David and Önal, Hüseyin C and Montanaro-Punzengruber, Jacqueline-Claire and Gassmann, Martin and Jonas, Peter M and Kulik, Akos and Bettler, Bernhard and Shigemoto, Ryuichi and Koppensteiner, Peter},
  issn         = {2050-084X},
  journal      = {eLife},
  publisher    = {eLife Sciences Publications},
  title        = {{GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals}},
  doi          = {10.7554/ELIFE.68274},
  volume       = {10},
  year         = {2021},
}

@article{9641,
  abstract     = {At the encounter with a novel environment, contextual memory formation is greatly enhanced, accompanied with increased arousal and active exploration. Although this phenomenon has been widely observed in animal and human daily life, how the novelty in the environment is detected and contributes to contextual memory formation has lately started to be unveiled. The hippocampus has been studied for many decades for its largely known roles in encoding spatial memory, and a growing body of evidence indicates a differential involvement of dorsal and ventral hippocampal divisions in novelty detection. In this brief review article, we discuss the recent findings of the role of mossy cells in the ventral hippocampal moiety in novelty detection and put them in perspective with other novelty-related pathways in the hippocampus. We propose a mechanism for novelty-driven memory acquisition in the dentate gyrus by the direct projection of ventral mossy cells to dorsal dentate granule cells. By this projection, the ventral hippocampus sends novelty signals to the dorsal hippocampus, opening a gate for memory encoding in dentate granule cells based on information coming from the entorhinal cortex. We conclude that, contrary to the presently accepted functional independence, the dorsal and ventral hippocampi cooperate to link the novelty and contextual information, and this dorso-ventral interaction is crucial for the novelty-dependent memory formation.},
  author       = {Fredes, Felipe and Shigemoto, Ryuichi},
  issn         = {10959564},
  journal      = {Neurobiology of Learning and Memory},
  publisher    = {Elsevier},
  title        = {{The role of hippocampal mossy cells in novelty detection}},
  doi          = {10.1016/j.nlm.2021.107486},
  volume       = {183},
  year         = {2021},
}

@inbook{9756,
  abstract     = {High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms.},
  author       = {Kaufmann, Walter and Kleindienst, David and Harada, Harumi and Shigemoto, Ryuichi},
  booktitle    = { Receptor and Ion Channel Detection in the Brain},
  isbn         = {9781071615218},
  keywords     = {Freeze-fracture replica: Deep learning, Immunogold labeling, Integral membrane protein, Electron microscopy},
  pages        = {267--283},
  publisher    = {Humana},
  title        = {{High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)}},
  doi          = {10.1007/978-1-0716-1522-5_19},
  volume       = {169},
  year         = {2021},
}

@article{8532,
  abstract     = {The molecular anatomy of synapses defines their characteristics in transmission and plasticity. Precise measurements of the number and distribution of synaptic proteins are important for our understanding of synapse heterogeneity within and between brain regions. Freeze–fracture replica immunogold electron microscopy enables us to analyze them quantitatively on a two-dimensional membrane surface. Here, we introduce Darea software, which utilizes deep learning for analysis of replica images and demonstrate its usefulness for quick measurements of the pre- and postsynaptic areas, density and distribution of gold particles at synapses in a reproducible manner. We used Darea for comparing glutamate receptor and calcium channel distributions between hippocampal CA3-CA1 spine synapses on apical and basal dendrites, which differ in signaling pathways involved in synaptic plasticity. We found that apical synapses express a higher density of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and a stronger increase of AMPA receptors with synaptic size, while basal synapses show a larger increase in N-methyl-D-aspartate (NMDA) receptors with size. Interestingly, AMPA and NMDA receptors are segregated within postsynaptic sites and negatively correlated in density among both apical and basal synapses. In the presynaptic sites, Cav2.1 voltage-gated calcium channels show similar densities in apical and basal synapses with distributions consistent with an exclusion zone model of calcium channel-release site topography.},
  author       = {Kleindienst, David and Montanaro-Punzengruber, Jacqueline-Claire and Bhandari, Pradeep and Case, Matthew J and Fukazawa, Yugo and Shigemoto, Ryuichi},
  issn         = {14220067},
  journal      = {International Journal of Molecular Sciences},
  number       = {18},
  publisher    = {MDPI},
  title        = {{Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses}},
  doi          = {10.3390/ijms21186737},
  volume       = {21},
  year         = {2020},
}

@article{7665,
  abstract     = {Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions.},
  author       = {Eguchi, Kohgaku and Velicky, Philipp and Hollergschwandtner, Elena and Itakura, Makoto and Fukazawa, Yugo and Danzl, Johann G and Shigemoto, Ryuichi},
  issn         = {16625102},
  journal      = {Frontiers in Cellular Neuroscience},
  publisher    = {Frontiers Media},
  title        = {{Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions}},
  doi          = {10.3389/fncel.2020.00063},
  volume       = {14},
  year         = {2020},
}

@article{6659,
  abstract     = {Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research.},
  author       = {Zenmyo, Naoki and Tokumaru, Hiroki and Uchinomiya, Shohei and Fuchida, Hirokazu and Tabata, Shigekazu and Hamachi, Itaru and Shigemoto, Ryuichi and Ojida, Akio},
  issn         = {00092673},
  journal      = {Bulletin of the Chemical Society of Japan},
  number       = {5},
  pages        = {995--1000},
  publisher    = {Bulletin of the Chemical Society of Japan},
  title        = {{Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins}},
  doi          = {10.1246/bcsj.20190034},
  volume       = {92},
  year         = {2019},
}

@article{7391,
  abstract     = {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.},
  author       = {Tabata, Shigekazu and Jevtic, Marijo and Kurashige, Nobutaka and Fuchida, Hirokazu and Kido, Munetsugu and Tani, Kazushi and Zenmyo, Naoki and Uchinomiya, Shohei and Harada, Harumi and Itakura, Makoto and Hamachi, Itaru and Shigemoto, Ryuichi and Ojida, Akio},
  issn         = {2589-0042},
  journal      = {iScience},
  number       = {12},
  pages        = {256--268},
  publisher    = {Elsevier},
  title        = {{Electron microscopic detection of single membrane proteins by a specific chemical labeling}},
  doi          = {10.1016/j.isci.2019.11.025},
  volume       = {22},
  year         = {2019},
}