TY - JOUR AB - The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny. AU - Cheung, Giselle T AU - Pauler, Florian AU - Koppensteiner, Peter AU - Krausgruber, Thomas AU - Streicher, Carmen AU - Schrammel, Martin AU - Özgen, Natalie Y AU - Ivec, Alexis AU - Bock, Christoph AU - Shigemoto, Ryuichi AU - Hippenmeyer, Simon ID - 12875 IS - 2 JF - Neuron SN - 0896-6273 TI - Multipotent progenitors instruct ontogeny of the superior colliculus VL - 112 ER - TY - JOUR AB - GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca 2+ -dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the “Flash and Freeze-fracture” method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals. AU - Koppensteiner, Peter AU - Bhandari, Pradeep AU - Önal, Hüseyin C AU - Borges Merjane, Carolina AU - Le Monnier, Elodie AU - Roy, Utsa AU - Nakamura, Yukihiro AU - Sadakata, Tetsushi AU - Sanbo, Makoto AU - Hirabayashi, Masumi AU - Rhee, JeongSeop AU - Brose, Nils AU - Jonas, Peter M AU - Shigemoto, Ryuichi ID - 15084 IS - 8 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles VL - 121 ER - TY - JOUR AB - The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission. AU - Chen, JingJing AU - Kaufmann, Walter AU - Chen, Chong AU - Arai, Itaru AU - Kim, Olena AU - Shigemoto, Ryuichi AU - Jonas, Peter M ID - 14843 JF - Neuron SN - 0896-6273 TI - Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse ER - TY - JOUR AB - Junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are specialized membrane contacts ubiquitous in eukaryotic cells. Concentration of intracellular signaling machinery near ER-PM junctions allows these domains to serve critical roles in lipid and Ca2+ signaling and homeostasis. Subcellular compartmentalization of protein kinase A (PKA) signaling also regulates essential cellular functions, however, no specific association between PKA and ER-PM junctional domains is known. Here, we show that in brain neurons type I PKA is directed to Kv2.1 channel-dependent ER-PM junctional domains via SPHKAP, a type I PKA-specific anchoring protein. SPHKAP association with type I PKA regulatory subunit RI and ER-resident VAP proteins results in the concentration of type I PKA between stacked ER cisternae associated with ER-PM junctions. This ER-associated PKA signalosome enables reciprocal regulation between PKA and Ca2+ signaling machinery to support Ca2+ influx and excitation-transcription coupling. These data reveal that neuronal ER-PM junctions support a receptor-independent form of PKA signaling driven by membrane depolarization and intracellular Ca2+, allowing conversion of information encoded in electrical signals into biochemical changes universally recognized throughout the cell. AU - Vierra, Nicholas C. AU - Ribeiro-Silva, Luisa AU - Kirmiz, Michael AU - Van Der List, Deborah AU - Bhandari, Pradeep AU - Mack, Olivia A. AU - Carroll, James AU - Le Monnier, Elodie AU - Aicher, Sue A. AU - Shigemoto, Ryuichi AU - Trimmer, James S. ID - 14253 JF - Nature Communications TI - Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling VL - 14 ER - TY - JOUR AB - 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. AU - Eguchi, Kohgaku AU - Le Monnier, Elodie AU - Shigemoto, Ryuichi ID - 13202 IS - 23 JF - The Journal of Neuroscience SN - 0270-6474 TI - Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons VL - 43 ER - TY - DATA AB - GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles at the presynaptic active zone (AZ). Strikingly, tonic and phasic release exhibit distinct coupling distances and are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. Double pre-embedding immunolabeling confirmed the co-localization of CAPS2 and SPO inside the same terminal. The cytosolic protein CAPS2 showed a synaptic vesicle (SV)-associated distribution similar to the vesicular transmembrane protein SPO. A newly developed “Flash and Freeze-fracture” method revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to a persistent RRP increase. Thus, we discovered structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals. AU - Shigemoto, Ryuichi ID - 13173 KW - medial habenula KW - GABAB receptor KW - vesicle release KW - Flash and Freeze KW - Flash and Freeze-fracture TI - Transition from tonic to phasic neurotransmitter release by presynaptic GABAB receptor activation in medial habenula terminals ER - TY - JOUR AB - 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. AU - Eguchi, Kohgaku AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Le Monnier, Elodie AU - Shigemoto, Ryuichi ID - 10890 JF - Frontiers in Neuroanatomy TI - The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals VL - 16 ER - TY - JOUR AB - 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. AU - Shigemoto, Ryuichi ID - 10889 IS - Supplement_1 JF - Microscopy SN - 2050-5698 TI - Electron microscopic visualization of single molecules by tag-mediated metal particle labeling VL - 71 ER - TY - JOUR AB - Alzheimer’s disease (AD) is characterized by a reorganization of brain activity determining network hyperexcitability and loss of synaptic plasticity. Precisely, a dysfunction in metabotropic GABAB receptor signalling through G protein-gated inwardly rectifying K+ (GIRK or Kir3) channels on the hippocampus has been postulated. Thus, we determined the impact of amyloid-β (Aβ) pathology in GIRK channel density, subcellular distribution, and its association with GABAB receptors in hippocampal CA1 pyramidal neurons from the APP/PS1 mouse model using quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL) and proximity ligation in situ assay (P-LISA). In wild type mice, single SDS-FRL detection revealed a similar dendritic gradient for GIRK1 and GIRK2 in CA1 pyramidal cells, with higher densities in spines, and GIRK3 showed a lower and uniform distribution. Double SDS-FRL showed a co-clustering of GIRK2 and GIRK1 in post- and presynaptic compartments, but not for GIRK2 and GIRK3. Likewise, double GABAB1 and GIRK2 SDS-FRL detection displayed a high degree of co-clustering in nanodomains (40–50 nm) mostly in spines and axon terminals. In APP/PS1 mice, the density of GIRK2 and GIRK1, but not for GIRK3, was significantly reduced along the neuronal surface of CA1 pyramidal cells and in axon terminals contacting them. Importantly, GABAB1 and GIRK2 co-clustering was not present in APP/PS1 mice. Similarly, P-LISA experiments revealed a significant reduction in GABAB1 and GIRK2 interaction on the hippocampus of this animal model. Overall, our results provide compelling evidence showing a significant reduction on the cell surface density of pre- and postsynaptic GIRK1 and GIRK2, but not GIRK3, and a decline in GABAB receptors and GIRK2 channels co-clustering in hippocampal pyramidal neurons from APP/PS1 mice, thus suggesting that a disruption in the GABAB receptor–GIRK channel membrane assembly causes dysregulation in the GABAB signalling via GIRK channels in this AD animal model. AU - Martín-Belmonte, Alejandro AU - Aguado, Carolina AU - Alfaro-Ruiz, Rocío AU - Moreno-Martínez, Ana Esther AU - de la Ossa, Luis AU - Aso, Ester AU - Gómez-Acero, Laura AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo AU - Ciruela, Francisco AU - Luján, Rafael ID - 12212 JF - Alzheimer's Research & Therapy KW - Cognitive Neuroscience KW - Neurology (clinical) KW - Neurology SN - 1758-9193 TI - Nanoscale alterations in GABAB receptors and GIRK channel organization on the hippocampus of APP/PS1 mice VL - 14 ER - TY - JOUR AB - 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. AU - Fredes Tolorza, Felipe A AU - Silva Sifuentes, Maria A AU - Koppensteiner, Peter AU - Kobayashi, Kenta AU - Jösch, Maximilian A AU - Shigemoto, Ryuichi ID - 7551 IS - 1 JF - Current Biology TI - Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation VL - 31 ER - TY - JOUR AB - 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. AU - Schöpf, Clemens L. AU - Ablinger, Cornelia AU - Geisler, Stefanie M. AU - Stanika, Ruslan I. AU - Campiglio, Marta AU - Kaufmann, Walter AU - Nimmervoll, Benedikt AU - Schlick, Bettina AU - Brockhaus, Johannes AU - Missler, Markus AU - Shigemoto, Ryuichi AU - Obermair, Gerald J. ID - 9330 IS - 14 JF - PNAS TI - Presynaptic α2δ subunits are key organizers of glutamatergic synapses VL - 118 ER - TY - JOUR AB - 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. AU - Fredes, Felipe AU - Shigemoto, Ryuichi ID - 9641 JF - Neurobiology of Learning and Memory SN - 10747427 TI - The role of hippocampal mossy cells in novelty detection VL - 183 ER - TY - JOUR AB - Rab-interacting molecule (RIM)-binding protein 2 (BP2) is a multidomain protein of the presynaptic active zone (AZ). By binding to RIM, bassoon (Bsn), and voltage-gated Ca2+ channels (CaV), it is considered to be a central organizer of the topography of CaV and release sites of synaptic vesicles (SVs) at the AZ. Here, we used RIM-BP2 knock-out (KO) mice and their wild-type (WT) littermates of either sex to investigate the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers (ANFs) with bushy cells (BCs) of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked EPSCs. Analysis of SV pool dynamics during high-frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by superresolution light and electron microscopy revealed an impaired topography of presynaptic CaV and a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in BCs of RIM-BP2-deficient mice in vivo. SIGNIFICANCE STATEMENT: Rab-interacting molecule (RIM)-binding proteins (BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmits auditory information at rates of up to hundreds of Hertz with submillisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+ influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of voltage-gated (CaV)2.1 Ca2+ channels, and fewer docked and membrane proximal synaptic vesicles (SVs). This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization. AU - Butola, Tanvi AU - Alvanos, Theocharis AU - Hintze, Anika AU - Koppensteiner, Peter AU - Kleindienst, David AU - Shigemoto, Ryuichi AU - Wichmann, Carolin AU - Moser, Tobias ID - 10051 IS - 37 JF - Journal of Neuroscience SN - 0270-6474 TI - RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse VL - 41 ER - TY - JOUR AB - Synaptic transmission, connectivity, and dendritic morphology mature in parallel during brain development and are often disrupted in neurodevelopmental disorders. Yet how these changes influence the neuronal computations necessary for normal brain function are not well understood. To identify cellular mechanisms underlying the maturation of synaptic integration in interneurons, we combined patch-clamp recordings of excitatory inputs in mouse cerebellar stellate cells (SCs), three-dimensional reconstruction of SC morphology with excitatory synapse location, and biophysical modeling. We found that postnatal maturation of postsynaptic strength was homogeneously reduced along the somatodendritic axis, but dendritic integration was always sublinear. However, dendritic branching increased without changes in synapse density, leading to a substantial gain in distal inputs. Thus, changes in synapse distribution, rather than dendrite cable properties, are the dominant mechanism underlying the maturation of neuronal computation. These mechanisms favor the emergence of a spatially compartmentalized two-stage integration model promoting location-dependent integration within dendritic subunits. AU - Biane, Celia AU - Rückerl, Florian AU - Abrahamsson, Therese AU - Saint-Cloment, Cécile AU - Mariani, Jean AU - Shigemoto, Ryuichi AU - Digregorio, David A. AU - Sherrard, Rachel M. AU - Cathala, Laurence ID - 10403 JF - eLife TI - Developmental emergence of two-stage nonlinear synaptic integration in cerebellar interneurons VL - 10 ER - TY - JOUR AB - 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. AU - Bhandari, Pradeep AU - Vandael, David H AU - Fernández-Fernández, Diego AU - Fritzius, Thorsten AU - Kleindienst, David AU - Önal, Hüseyin C AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Gassmann, Martin AU - Jonas, Peter M AU - Kulik, Akos AU - Bettler, Bernhard AU - Shigemoto, Ryuichi AU - Koppensteiner, Peter ID - 9437 JF - eLife TI - GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals VL - 10 ER - TY - CHAP AB - 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. AU - Kaufmann, Walter AU - Kleindienst, David AU - Harada, Harumi AU - Shigemoto, Ryuichi ID - 9756 KW - Freeze-fracture replica: Deep learning KW - Immunogold labeling KW - Integral membrane protein KW - Electron microscopy SN - 9781071615218 T2 - Receptor and Ion Channel Detection in the Brain TI - High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL) VL - 169 ER - TY - JOUR AB - In the cerebellum, GluD2 is exclusively expressed in Purkinje cells, where it regulates synapse formation and regeneration, synaptic plasticity, and motor learning. Delayed cognitive development in humans with GluD2 gene mutations suggests extracerebellar functions of GluD2. However, extracerebellar expression of GluD2 and its relationship with that of GluD1 are poorly understood. GluD2 mRNA and protein were widely detected, with relatively high levels observed in the olfactory glomerular layer, medial prefrontal cortex, cingulate cortex, retrosplenial granular cortex, olfactory tubercle, subiculum, striatum, lateral septum, anterodorsal thalamic nucleus, and arcuate hypothalamic nucleus. These regions were also enriched for GluD1, and many individual neurons coexpressed the two GluDs. In the retrosplenial granular cortex, GluD1 and GluD2 were selectively expressed at PSD‐95‐expressing glutamatergic synapses, and their coexpression on the same synapses was shown by SDS‐digested freeze‐fracture replica labeling. Biochemically, GluD1 and GluD2 formed coimmunoprecipitable complex formation in HEK293T cells and in the cerebral cortex and hippocampus. We further estimated the relative protein amount by quantitative immunoblotting using GluA2/GluD2 and GluA2/GluD1 chimeric proteins as standards for titration of GluD1 and GluD2 antibodies. Intriguingly, the relative amount of GluD2 was almost comparable to that of GluD1 in the postsynaptic density fraction prepared from the cerebral cortex and hippocampus. In contrast, GluD2 was overwhelmingly predominant in the cerebellum. Thus, we have determined the relative extracerebellar expression of GluD1 and GluD2 at regional, neuronal, and synaptic levels. These data provide a molecular–anatomical basis for possible competitive and cooperative interactions of GluD family members at synapses in various brain regions. AU - Nakamoto, Chihiro AU - Konno, Kohtarou AU - Miyazaki, Taisuke AU - Nakatsukasa, Ena AU - Natsume, Rie AU - Abe, Manabu AU - Kawamura, Meiko AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi AU - Yamasaki, Miwako AU - Sakimura, Kenji AU - Watanabe, Masahiko ID - 7148 IS - 6 JF - Journal of Comparative Neurology SN - 0021-9967 TI - Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain VL - 528 ER - TY - JOUR AB - Metabotropic γ-aminobutyric acid (GABAB) receptors contribute to the control of network activity and information processing in hippocampal circuits by regulating neuronal excitability and synaptic transmission. The dysfunction in the dentate gyrus (DG) has been implicated in Alzheimer´s disease (AD). Given the involvement of GABAB receptors in AD, to determine their subcellular localisation and possible alteration in granule cells of the DG in a mouse model of AD at 12 months of age, we used high-resolution immunoelectron microscopic analysis. Immunohistochemistry at the light microscopic level showed that the regional and cellular expression pattern of GABAB1 was similar in an AD model mouse expressing mutated human amyloid precursor protein and presenilin1 (APP/PS1) and in age-matched wild type mice. High-resolution immunoelectron microscopy revealed a distance-dependent gradient of immunolabelling for GABAB receptors, increasing from proximal to distal dendrites in both wild type and APP/PS1 mice. However, the overall density of GABAB receptors at the neuronal surface of these postsynaptic compartments of granule cells was significantly reduced in APP/PS1 mice. Parallel to this reduction in surface receptors, we found a significant increase in GABAB1 at cytoplasmic sites. GABAB receptors were also detected at presynaptic sites in the molecular layer of the DG. We also found a decrease in plasma membrane GABAB receptors in axon terminals contacting dendritic spines of granule cells, which was more pronounced in the outer than in the inner molecular layer. Altogether, our data showing post- and presynaptic reduction in surface GABAB receptors in the DG suggest the alteration of the GABAB-mediated modulation of excitability and synaptic transmission in granule cells, which may contribute to the cognitive dysfunctions in the APP/PS1 model of AD AU - Martín-Belmonte, Alejandro AU - Aguado, Carolina AU - Alfaro-Ruíz, Rocío AU - Moreno-Martínez, Ana Esther AU - De La Ossa, Luis AU - Martínez-Hernández, José AU - Buisson, Alain AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo AU - Luján, Rafael ID - 7664 IS - 7 JF - International journal of molecular sciences TI - Density of GABAB receptors is reduced in granule cells of the hippocampus in a mouse model of Alzheimer's disease VL - 21 ER - TY - JOUR AB - 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. AU - Eguchi, Kohgaku AU - Velicky, Philipp AU - Hollergschwandtner, Elena AU - Itakura, Makoto AU - Fukazawa, Yugo AU - Danzl, Johann G AU - Shigemoto, Ryuichi ID - 7665 JF - Frontiers in Cellular Neuroscience SN - 16625102 TI - Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions VL - 14 ER - TY - JOUR AB - Type 1 metabotropic glutamate receptors (mGluR1s) are key elements in neuronal signaling. While their function is well documented in slices, requirements for their activation in vivo are poorly understood. We examine this question in adult mice in vivo using 2-photon imaging of cerebellar molecular layer interneurons (MLIs) expressing GCaMP. In anesthetized mice, parallel fiber activation evokes beam-like Cai rises in postsynaptic MLIs which depend on co-activation of mGluR1s and ionotropic glutamate receptors (iGluRs). In awake mice, blocking mGluR1 decreases Cai rises associated with locomotion. In vitro studies and freeze-fracture electron microscopy show that the iGluR-mGluR1 interaction is synergistic and favored by close association of the two classes of receptors. Altogether our results suggest that mGluR1s, acting in synergy with iGluRs, potently contribute to processing cerebellar neuronal signaling under physiological conditions. AU - Bao, Jin AU - Graupner, Michael AU - Astorga, Guadalupe AU - Collin, Thibault AU - Jalil, Abdelali AU - Indriati, Dwi Wahyu AU - Bradley, Jonathan AU - Shigemoto, Ryuichi AU - Llano, Isabel ID - 7878 JF - eLife TI - Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo VL - 9 ER - TY - JOUR AB - The hippocampus plays key roles in learning and memory and is a main target of Alzheimer's disease (AD), which causes progressive memory impairments. Despite numerous investigations about the processes required for the normal hippocampal functions, the neurotransmitter receptors involved in the synaptic deficits by which AD disables the hippocampus are not yet characterized. By combining histoblots, western blots, immunohistochemistry and high‐resolution immunoelectron microscopic methods for GABAB receptors, this study provides a quantitative description of the expression and the subcellular localization of GABAB1 in the hippocampus in a mouse model of AD at 1, 6 and 12 months of age. Western blots and histoblots showed that the total amount of protein and the laminar expression pattern of GABAB1 were similar in APP/PS1 mice and in age‐matched wild‐type mice. In contrast, immunoelectron microscopic techniques showed that the subcellular localization of GABAB1 subunit did not change significantly in APP/PS1 mice at 1 month of age, was significantly reduced in the stratum lacunosum‐moleculare of CA1 pyramidal cells at 6 months of age and significantly reduced at the membrane surface of CA1 pyramidal cells at 12 months of age. This reduction of plasma membrane GABAB1 was paralleled by a significant increase of the subunit at the intracellular sites. We further observed a decrease of membrane‐targeted GABAB receptors in axon terminals contacting CA1 pyramidal cells. Our data demonstrate compartment‐ and age‐dependent reduction of plasma membrane‐targeted GABAB receptors in the CA1 region of the hippocampus, suggesting that this decrease might be enough to alter the GABAB‐mediated synaptic transmission taking place in AD. AU - Martín-Belmonte, Alejandro AU - Aguado, Carolina AU - Alfaro-Ruíz, Rocío AU - Moreno-Martínez, Ana Esther AU - De La Ossa, Luis AU - Martínez-Hernández, José AU - Buisson, Alain AU - Früh, Simon AU - Bettler, Bernhard AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo AU - Luján, Rafael ID - 7207 IS - 3 JF - Brain Pathology SN - 10156305 TI - Reduction in the neuronal surface of post and presynaptic GABA>B< receptors in the hippocampus in a mouse model of Alzheimer's disease VL - 30 ER - TY - JOUR AB - 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. AU - Kleindienst, David AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Bhandari, Pradeep AU - Case, Matthew J AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi ID - 8532 IS - 18 JF - International Journal of Molecular Sciences SN - 16616596 TI - Deep learning-assisted high-throughput analysis of freeze-fracture replica images applied to glutamate receptors and calcium channels at hippocampal synapses VL - 21 ER - TY - JOUR AB - 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. AU - Zenmyo, Naoki AU - Tokumaru, Hiroki AU - Uchinomiya, Shohei AU - Fuchida, Hirokazu AU - Tabata, Shigekazu AU - Hamachi, Itaru AU - Shigemoto, Ryuichi AU - Ojida, Akio ID - 6659 IS - 5 JF - Bulletin of the Chemical Society of Japan SN - 00092673 TI - Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins VL - 92 ER - TY - JOUR AB - Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at the subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can be bidirectionally modulated by a HCN channel blocker, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mouse cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 mM; estimated endogenous cAMP concentration 13 mM). In addition, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily by altering the resting membrane potential and are associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators with an opportunity to finely tune energy consumption and temporal delays across axons in the brain. AU - Byczkowicz, Niklas AU - Eshra, Abdelmoneim AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Trevisiol, Andrea AU - Hirrlinger, Johannes AU - Kole, Maarten Hp AU - Shigemoto, Ryuichi AU - Hallermann, Stefan ID - 6868 JF - eLife TI - HCN channel-mediated neuromodulation can control action potential velocity and fidelity in central axons VL - 8 ER - TY - JOUR AU - Kasugai, Yu AU - Vogel, Elisabeth AU - Hörtnagl, Heide AU - Schönherr, Sabine AU - Paradiso, Enrica AU - Hauschild, Markus AU - Göbel, Georg AU - Milenkovic, Ivan AU - Peterschmitt, Yvan AU - Tasan, Ramon AU - Sperk, Günther AU - Shigemoto, Ryuichi AU - Sieghart, Werner AU - Singewald, Nicolas AU - Lüthi, Andreas AU - Ferraguti, Francesco ID - 7099 IS - 4 JF - Neuron SN - 0896-6273 TI - Structural and functional remodeling of amygdala GABAergic synapses in associative fear learning VL - 104 ER - TY - JOUR AB - Glutamate is the major excitatory neurotransmitter in the CNS binding to a variety of glutamate receptors. Metabotropic glutamate receptors (mGluR1 to mGluR8) can act excitatory or inhibitory, depending on associated signal cascades. Expression and localization of inhibitory acting mGluRs at inner hair cells (IHCs) in the cochlea are largely unknown. Here, we analyzed expression of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8 and investigated their localization with respect to the presynaptic ribbon of IHC synapses. We detected transcripts for mGluR2, mGluR3, and mGluR4 as well as for mGluR7a, mGluR7b, mGluR8a, and mGluR8b splice variants. Using receptor-specific antibodies in cochlear wholemounts, we found expression of mGluR2, mGluR4, and mGluR8b close to presynaptic ribbons. Super resolution and confocal microscopy in combination with 3-dimensional reconstructions indicated a postsynaptic localization of mGluR2 that overlaps with postsynaptic density protein 95 on dendrites of afferent type I spiral ganglion neurons. In contrast, mGluR4 and mGluR8b were expressed at the presynapse close to IHC ribbons. In summary, we localized in detail 3 mGluR types at IHC ribbon synapses, providing a fundament for new therapeutical strategies that could protect the cochlea against noxious stimuli and excitotoxicity. AU - Klotz, Lisa AU - Wendler, Olaf AU - Frischknecht, Renato AU - Shigemoto, Ryuichi AU - Schulze, Holger AU - Enz, Ralf ID - 7179 IS - 12 JF - FASEB Journal TI - Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses VL - 33 ER - 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 - 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 - TY - JOUR AB - Metabotropic GABAB receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABAB receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABAB1 was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABAB receptors with two key effector ion channels, the G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel and the voltage-dependent Ca2+ channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABAB receptors co-assembled with GIRK and CaV2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABAB1 and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABAB1 and CaV2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABAB1 and GIRK2 or CaV2.1 channels was detected, inter-cluster distance for GABAB1 and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABAB1 and CaV2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABAB receptors are associated with GIRK and CaV2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABAB receptors and their effector ion channels in the cerebellar network. AU - Luján, Rafael AU - Aguado, Carolina AU - Ciruela, Francisco AU - Cózar, Javier AU - Kleindienst, David AU - De La Ossa, Luis AU - Bettler, Bernhard AU - Wickman, Kevin AU - Watanabe, Masahiko AU - Shigemoto, Ryuichi AU - Fukazawa, Yugo ID - 612 IS - 3 JF - Brain Structure and Function TI - Differential association of GABAB receptors with their effector ion channels in Purkinje cells VL - 223 ER - TY - JOUR AB - Many central synapses contain a single presynaptic active zone and a single postsynaptic density. Vesicular release statistics at such “simple synapses” indicate that they contain a small complement of docking sites where vesicles repetitively dock and fuse. In this work, we investigate functional and morphological aspects of docking sites at simple synapses made between cerebellar parallel fibers and molecular layer interneurons. Using immunogold labeling of SDS-treated freeze-fracture replicas, we find that Cav2.1 channels form several clusters per active zone with about nine channels per cluster. The mean value and range of intersynaptic variation are similar for Cav2.1 cluster numbers and for functional estimates of docking-site numbers obtained from the maximum numbers of released vesicles per action potential. Both numbers grow in relation with synaptic size and decrease by a similar extent with age between 2 wk and 4 wk postnatal. Thus, the mean docking-site numbers were 3.15 at 2 wk (range: 1–10) and 2.03 at 4 wk (range: 1–4), whereas the mean numbers of Cav2.1 clusters were 2.84 at 2 wk (range: 1–8) and 2.37 at 4 wk (range: 1–5). These changes were accompanied by decreases of miniature current amplitude (from 93 pA to 56 pA), active-zone surface area (from 0.0427 μm2 to 0.0234 μm2), and initial success rate (from 0.609 to 0.353), indicating a tightening of synaptic transmission with development. Altogether, these results suggest a close correspondence between the number of functionally defined vesicular docking sites and that of clusters of voltage-gated calcium channels. AU - Miki, Takafumi AU - Kaufmann, Walter AU - Malagon, Gerardo AU - Gomez, Laura AU - Tabuchi, Katsuhiko AU - Watanabe, Masahiko AU - Shigemoto, Ryuichi AU - Marty, Alain ID - 693 IS - 26 JF - PNAS SN - 00278424 TI - Numbers of presynaptic Ca2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses VL - 114 ER - TY - JOUR AB - The neurotransmitter receptor subtype, number, density, and distribution relative to the location of transmitter release sites are key determinants of signal transmission. AMPA-type ionotropic glutamate receptors (AMPARs) containing GluA3 and GluA4 subunits are prominently expressed in subsets of neurons capable of firing action potentials at high frequencies, such as auditory relay neurons. The auditory nerve (AN) forms glutamatergic synapses on two types of relay neurons, bushy cells (BCs) and fusiform cells (FCs) of the cochlear nucleus. AN-BC and AN-FC synapses have distinct kinetics; thus, we investigated whether the number, density, and localization of GluA3 and GluA4 subunits in these synapses are differentially organized using quantitative freeze-fracture replica immunogold labeling. We identify a positive correlation between the number of AMPARs and the size of AN-BC and AN-FC synapses. Both types of AN synapses have similar numbers of AMPARs; however, the AN-BC have a higher density of AMPARs than AN-FC synapses, because the AN-BC synapses are smaller. A higher number and density of GluA3 subunits are observed at AN-BC synapses, whereas a higher number and density of GluA4 subunits are observed at AN-FC synapses. The intrasynaptic distribution of immunogold labeling revealed that AMPAR subunits, particularly GluA3, are concentrated at the center of the AN-BC synapses. The central distribution of AMPARs is absent in GluA3-knockout mice, and gold particles are evenly distributed along the postsynaptic density. GluA4 gold labeling was homogenously distributed along both synapse types. Thus, GluA3 and GluA4 subunits are distributed at AN synapses in a target-cell-dependent manner. AU - Rubio, María AU - Matsui, Ko AU - Fukazawa, Yugo AU - Kamasawa, Naomi AU - Harada, Harumi AU - Itakura, Makoto AU - Molnár, Elek AU - Abe, Manabu AU - Sakimura, Kenji AU - Shigemoto, Ryuichi ID - 736 IS - 8 JF - Brain Structure and Function SN - 18632653 TI - The number and distribution of AMPA receptor channels containing fast kinetic GluA3 and GluA4 subunits at auditory nerve synapses depend on the target cells VL - 222 ER - TY - JOUR AB - Developments in bioengineering and molecular biology have introduced a palette of genetically encoded probes for identification of specific cell populations in electron microscopy. These probes can be targeted to distinct cellular compartments, rendering them electron dense through a subsequent chemical reaction. These electron densities strongly increase the local contrast in samples prepared for electron microscopy, allowing three major advances in ultrastructural mapping of circuits: genetic identification of circuit components, targeted imaging of regions of interest and automated analysis of the tagged circuits. Together, the gains from these advances can decrease the time required for the analysis of targeted circuit motifs by over two orders of magnitude. These genetic encoded tags for electron microscopy promise to simplify the analysis of circuit motifs and become a central tool for structure‐function studies of synaptic connections in the brain. We review the current state‐of‐the‐art with an emphasis on connectomics, the quantitative analysis of neuronal structures and motifs. AU - Shigemoto, Ryuichi AU - Jösch, Maximilian A ID - 740 IS - 6 JF - WIREs Developmental Biology SN - 17597684 TI - The genetic encoded toolbox for electron microscopy and connectomics VL - 6 ER - TY - JOUR AB - Metabotropic glutamate receptor subtype 5 (mGluR5) is crucially implicated in the pathophysiology of Fragile X Syndrome (FXS); however, its dysfunction at the sub-cellular level, and related synaptic and cognitive phenotypes are unexplored. Here, we probed the consequences of mGluR5/Homer scaffold disruption for mGluR5 cell-surface mobility, synaptic N-methyl-D-Aspartate receptor (NMDAR) function, and behavioral phenotypes in the second-generation Fmr1 knockout (KO) mouse. Using single-molecule tracking, we found that mGluR5 was significantly more mobile at synapses in hippocampal Fmr1 KO neurons, causing an increased synaptic surface co-clustering of mGluR5 and NMDAR. This correlated with a reduced amplitude of synaptic NMDAR currents, a lack of their mGluR5-Activated long-Term depression, and NMDAR/hippocampus dependent cognitive deficits. These synaptic and behavioral phenomena were reversed by knocking down Homer1a in Fmr1 KO mice. Our study provides a mechanistic link between changes of mGluR5 dynamics and pathological phenotypes of FXS, unveiling novel targets for mGluR5-based therapeutics. AU - Aloisi, Elisabetta AU - Le Corf, Katy AU - Dupuis, Julien AU - Zhang, Pei AU - Ginger, Melanie AU - Labrousse, Virginie AU - Spatuzza, Michela AU - Georg Haberl, Matthias AU - Costa, Lara AU - Shigemoto, Ryuichi AU - Tappe Theodor, Anke AU - Drago, Fillippo AU - Vincenzo Piazza, Pier AU - Mulle, Christophe AU - Groc, Laurent AU - Ciranna, Lucia AU - Catania, Maria AU - Frick, Andreas ID - 746 IS - 1 JF - Nature Communications SN - 20411723 TI - Altered surface mGluR5 dynamics provoke synaptic NMDAR dysfunction and cognitive defects in Fmr1 knockout mice VL - 8 ER - TY - JOUR AB - Left-right asymmetry is a fundamental feature of higher-order brain structure; however, the molecular basis of brain asymmetry remains unclear. We recently identified structural and functional asymmetries in mouse hippocampal circuitry that result from the asymmetrical distribution of two distinct populations of pyramidal cell synapses that differ in the density of the NMDA receptor subunit GluRε2 (also known as NR2B, GRIN2B or GluN2B). By examining the synaptic distribution of ε2 subunits, we previously found that β2-microglobulin-deficient mice, which lack cell surface expression of the vast majority of major histocompatibility complex class I (MHCI) proteins, do not exhibit circuit asymmetry. In the present study, we conducted electrophysiological and anatomical analyses on the hippocampal circuitry of mice with a knockout of the paired immunoglobulin-like receptor B (PirB), an MHCI receptor. As in β2-microglobulin-deficient mice, the PirB-deficient hippocampus lacked circuit asymmetries. This finding that MHCI loss-of-function mice and PirB knockout mice have identical phenotypes suggests that MHCI signals that produce hippocampal asymmetries are transduced through PirB. Our results provide evidence for a critical role of the MHCI/PirB signaling system in the generation of asymmetries in hippocampal circuitry. AU - Ukai, Hikari AU - Kawahara, Aiko AU - Hirayama, Keiko AU - Case, Matthew J AU - Aino, Shotaro AU - Miyabe, Masahiro AU - Wakita, Ken AU - Oogi, Ryohei AU - Kasayuki, Michiyo AU - Kawashima, Shihomi AU - Sugimoto, Shunichi AU - Chikamatsu, Kanako AU - Nitta, Noritaka AU - Koga, Tsuneyuki AU - Shigemoto, Ryuichi AU - Takai, Toshiyuki AU - Ito, Isao ID - 682 IS - 6 JF - PLoS One SN - 19326203 TI - PirB regulates asymmetries in hippocampal circuitry VL - 12 ER - TY - JOUR AB - Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABABreceptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins. AU - Booker, Sam AU - Althof, Daniel AU - Gross, Anna AU - Loreth, Desiree AU - Müller, Johanna AU - Unger, Andreas AU - Fakler, Bernd AU - Varro, Andrea AU - Watanabe, Masahiko AU - Gassmann, Martin AU - Bettler, Bernhard AU - Shigemoto, Ryuichi AU - Vida, Imre AU - Kulik, Ákos ID - 1083 IS - 3 JF - Cerebral Cortex TI - KCTD12 auxiliary proteins modulate kinetics of GABAB receptor-mediated inhibition in Cholecystokinin-containing interneurons VL - 27 ER - TY - JOUR AB - Adaptations of vestibulo-ocular and optokinetic response eye movements have been studied as an experimental model of cerebellum-dependent motor learning. Several previous physiological and pharmacological studies have consistently suggested that the cerebellar flocculus (FL) Purkinje cells (P-cells) and the medial vestibular nucleus (MVN) neurons targeted by FL (FL-targeted MVN neurons) may respectively maintain the memory traces of short- and long-term adaptation. To study the basic structures of the FL-MVN synapses by light microscopy (LM) and electron microscopy (EM), we injected green florescence protein (GFP)-expressing lentivirus into FL to anterogradely label the FL P-cell axons in C57BL/6J mice. The FL P-cell axonal boutons were distributed in the magnocellular MVN and in the border region of parvocellular MVN and prepositus hypoglossi (PrH). In the magnocellular MVN, the FL-P cell axons mainly terminated on somata and proximal dendrites. On the other hand, in the parvocellular MVN/PrH, the FL P-cell axonal synaptic boutons mainly terminated on the relatively small-diameter (< 1 μm) distal dendrites of MVN neurons, forming symmetrical synapses. The majority of such parvocellular MVN/PrH neurons were determined to be glutamatergic by immunocytochemistry and in-situ hybridization of GFP expressing transgenic mice. To further examine the spatial relationship between the synapses of FL P-cells and those of vestibular nerve on the neurons of the parvocellular MVN/ PrH, we added injections of biotinylated dextran amine into the semicircular canal and anterogradely labeled vestibular nerve axons in some mice. The MVN dendrites receiving the FL P-cell axonal synaptic boutons often closely apposed vestibular nerve synaptic boutons in both LM and EM studies. Such a partial overlap of synaptic boutons of FL P-cell axons with those of vestibular nerve axons in the distal dendrites of MVN neurons suggests that inhibitory synapses of FL P-cells may influence the function of neighboring excitatory synapses of vestibular nerve in the parvocellular MVN/PrH neurons. AU - Matsuno, Hitomi AU - Kudoh, Moeko AU - Watakabe, Akiya AU - Yamamori, Tetsuo AU - Shigemoto, Ryuichi AU - Nagao, Soichi ID - 1278 IS - 10 JF - PLoS One TI - Distribution and structure of synapses on medial vestibular nuclear neurons targeted by cerebellar flocculus purkinje cells and vestibular nerve in mice: Light and electron microscopy studies VL - 11 ER - TY - CHAP AB - Immunogold labeling of freeze-fracture replicas has recently been used for high-resolution visualization of protein localization in electron microscopy. This method has higher labeling efficiency than conventional immunogold methods for membrane molecules allowing precise quantitative measurements. However, one of the limitations of freeze-fracture replica immunolabeling is difficulty in keeping structural orientation and identifying labeled profiles in complex tissues like brain. The difficulty is partly due to fragmentation of freeze-fracture replica preparations during labeling procedures and limited morphological clues on the replica surface. To overcome these issues, we introduce here a grid-glued replica method combined with SEM observation. This method allows histological staining before dissolving the tissue and easy handling of replicas during immunogold labeling, and keeps the whole replica surface intact without fragmentation. The procedure described here is also useful for matched double-replica analysis allowing further identification of labeled profiles in corresponding P-face and E-face. AU - Harada, Harumi AU - Shigemoto, Ryuichi ID - 1094 SN - 0302-9743 T2 - High-Resolution Imaging of Cellular Proteins TI - Immunogold protein localization on grid-glued freeze-fracture replicas VL - 1474 ER - TY - JOUR AB - Type 1 metabotropic glutamate (mGlu1) receptors play a pivotal role in different forms of synaptic plasticity in the cerebellar cortex, e.g. long-term depression at glutamatergic synapses and rebound potentiation at GABAergic synapses. These various forms of plasticity might depend on the subsynaptic arrangement of the receptor in Purkinje cells that can be regulated by protein-protein interactions. This study investigated, by means of the freeze-fracture replica immunogold labelling method, the subcellular localization of mGlu1 receptors in the rodent cerebellum and whether Homer proteins regulate their subsynaptic distribution. We observed a widespread extrasynaptic localization of mGlu1 receptors and confirmed their peri-synaptic enrichment at glutamatergic synapses. Conversely, we detected mGlu1 receptors within the main body of GABAergic synapses onto Purkinje cell dendrites. Although Homer proteins are known to interact with the mGlu1 receptor C-terminus, we could not detect Homer3, the most abundant Homer protein in the cerebellar cortex, at GABAergic synapses by pre-embedding and post-embedding immunoelectron microscopy. We then hypothesized a critical role for Homer proteins in the peri-junctional localization of mGlu1 receptors at glutamatergic synapses. To disrupt Homer-associated protein complexes, mice were tail-vein injected with the membrane-permeable dominant-negative TAT-Homer1a. Freeze-fracture replica immunogold labelling analysis showed no significant alteration in the mGlu1 receptor distribution pattern at parallel fibre-Purkinje cell synapses, suggesting that other scaffolding proteins are involved in the peri-synaptic confinement. The identification of interactors that regulate the subsynaptic localization of the mGlu1 receptor at neurochemically distinct synapses may offer new insight into its trafficking and intracellular signalling. AU - Mansouri, Mahnaz AU - Kasugai, Yu AU - Fukazawa, Yugo AU - Bertaso, Federica AU - Raynaud, Fabrice AU - Perroy, Julie AU - Fagni, Laurent AU - Walter Kaufmann AU - Watanabe, Masahiko AU - Ryuichi Shigemoto AU - Ferraguti, Francesco ID - 1515 IS - 2 JF - European Journal of Neuroscience TI - Distinct subsynaptic localization of type 1 metabotropic glutamate receptors at glutamatergic and GABAergic synapses in the rodent cerebellar cortex VL - 41 ER - TY - JOUR AB - Endocannabinoids (eCBs) play key roles in brain function, acting as modulatory signals in synaptic transmission and plasticity. They are recognized as retrograde messengers that mediate long-term synaptic depression (LTD), but their ability to induce long-term potentiation (LTP) is poorly known. We show that eCBs induce the long-term enhancement of transmitter release at single hippocampal synapses through stimulation of astrocytes when coincident with postsynaptic activity. This LTP requires the coordinated activity of the 3 elements of the tripartite synapse: 1) eCB-evoked astrocyte calcium signal that stimulates glutamate release; 2) postsynaptic nitric oxide production; and 3) activation of protein kinase C and presynaptic group I metabotropic glutamate receptors, whose location at presynaptic sites was confirmed by immunoelectron microscopy. Hence, while eCBs act as retrograde signals to depress homoneuronal synapses, they serve as lateral messengers to induce LTP in distant heteroneuronal synapses through stimulation of astrocytes. Therefore, eCBs can trigger LTP through stimulation of astrocyte-neuron signaling, revealing novel cellular mechanisms of eCB effects on synaptic plasticity. AU - Gómez-Gonzalo, Marta AU - Navarrete, Marta AU - Perea, Gertrudis AU - Covelo, Ana AU - Martín-Fernández, Mario AU - Ryuichi Shigemoto AU - Luján, Rafael AU - Araque, Alfonso ID - 1514 IS - 10 JF - Cerebral Cortex TI - Endocannabinoids induce lateral long term potentiation of transmitter release by stimulation of gliotransmission VL - 25 ER - TY - JOUR AB - Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca2+ channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca2+] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca2+ buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca2+ sensors for vesicular release are located at the perimeter of VGCC clusters (<30nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course. AU - Nakamura, Yukihiro AU - Harada, Harumi AU - Kamasawa, Naomi AU - Matsui, Ko AU - Rothman, Jason AU - Shigemoto, Ryuichi AU - Silver, R Angus AU - Digregorio, David AU - Takahashi, Tomoyuki ID - 1546 IS - 1 JF - Neuron TI - Nanoscale distribution of presynaptic Ca2+ channels and its impact on vesicular release during development VL - 85 ER - TY - JOUR AB - γ-Aminobutyric acid (GABA)- and glycine-mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium-chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high-resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3-subunit of GABAA receptors and KCC2, GABAA receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABAA and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor-immunoreactive dendrites, we found that gephyrin-immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4-10-μm-long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABAA and glycine receptors along the dendrites of spinal neurons. AU - Javdani, Fariba AU - Holló, Krisztina AU - Hegedűs, Krisztina AU - Kis, Gréta AU - Hegyi, Zoltán AU - Dócs, Klaudia AU - Kasugai, Yu AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi AU - Antal, Miklós ID - 1557 IS - 13 JF - Journal of Comparative Neurology TI - Differential expression patterns of K+Cl- cotransporter 2 in neurons within the superficial spinal dorsal horn of rats VL - 523 ER - TY - JOUR AB - Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wildtype mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation. AU - Edamura, Mitsuhiro AU - Murakami, Gen AU - Meng, Hongrui AU - Itakura, Makoto AU - Shigemoto, Ryuichi AU - Fukuda, Atsuo AU - Nakahara, Daiichiro ID - 1895 IS - 9 JF - PLoS One TI - Functional deficiency of MHC class i enhances LTP and abolishes LTD in the nucleus accumbens of mice VL - 9 ER - TY - JOUR AB - Fast synaptic transmission is important for rapid information processing. To explore the maximal rate of neuronal signaling and to analyze the presynaptic mechanisms, we focused on the input layer of the cerebellar cortex, where exceptionally high action potential (AP) frequencies have been reported invivo. With paired recordings between presynaptic cerebellar mossy fiber boutons and postsynaptic granule cells, we demonstrate reliable neurotransmission upto ~1 kHz. Presynaptic APs are ultrafast, with ~100μs half-duration. Both Kv1 and Kv3 potassium channels mediate the fast repolarization, rapidly inactivating sodium channels ensure metabolic efficiency, and little AP broadening occurs during bursts of up to 1.5 kHz. Presynaptic Cav2.1 (P/Q-type) calcium channels open efficiently during ultrafast APs. Furthermore, a subset of synaptic vesicles is tightly coupled to Ca2+ channels, and vesicles are rapidly recruited to the release site. These data reveal mechanisms of presynaptic AP generation and transmitter release underlying neuronal kHz signaling. AU - Ritzau Jost, Andreas AU - Delvendahl, Igor AU - Rings, Annika AU - Byczkowicz, Niklas AU - Harada, Harumi AU - Shigemoto, Ryuichi AU - Hirrlinger, Johannes AU - Eilers, Jens AU - Hallermann, Stefan ID - 1898 IS - 1 JF - Neuron TI - Ultrafast action potentials mediate kilohertz signaling at a central synapse VL - 84 ER - TY - JOUR AB - Cerebellar motor learning is suggested to be caused by long-term plasticity of excitatory parallel fiber-Purkinje cell (PF-PC) synapses associated with changes in the number of synaptic AMPA-type glutamate receptors (AMPARs). However, whether the AMPARs decrease or increase in individual PF-PC synapses occurs in physiological motor learning and accounts for memory that lasts over days remains elusive. We combined quantitative SDS-digested freeze-fracture replica labeling for AMPAR and physical dissector electron microscopy with a simple model of cerebellar motor learning, adaptation of horizontal optokinetic response (HOKR) in mouse. After 1-h training of HOKR, short-term adaptation (STA) was accompanied with transient decrease in AMPARs by 28% in target PF-PC synapses. STA was well correlated with AMPAR decrease in individual animals and both STA and AMPAR decrease recovered to basal levels within 24 h. Surprisingly, long-termadaptation (LTA) after five consecutive daily trainings of 1-h HOKR did not alter the number of AMPARs in PF-PC synapses but caused gradual and persistent synapse elimination by 45%, with corresponding PC spine loss by the fifth training day. Furthermore, recovery of LTA after 2 wk was well correlated with increase of PF-PC synapses to the control level. Our findings indicate that the AMPARs decrease in PF-PC synapses and the elimination of these synapses are in vivo engrams in short- and long-term motor learning, respectively, showing a unique type of synaptic plasticity that may contribute to memory consolidation. AU - Wang, Wen AU - Nakadate, Kazuhiko AU - Masugi Tokita, Miwako AU - Shutoh, Fumihiro AU - Aziz, Wajeeha AU - Tarusawa, Etsuko AU - Lörincz, Andrea AU - Molnár, Elek AU - Kesaf, Sebnem AU - Li, Yunqing AU - Fukazawa, Yugo AU - Nagao, Soichi AU - Shigemoto, Ryuichi ID - 1920 IS - 1 JF - PNAS TI - Distinct cerebellar engrams in short-term and long-term motor learning VL - 111 ER - TY - JOUR AB - Long-lasting memories are formed when the stimulus is temporally distributed (spacing effect). However, the synaptic mechanisms underlying this robust phenomenon and the precise time course of the synaptic modifications that occur during learning remain unclear. Here we examined the adaptation of horizontal optokinetic response in mice that underwent 1 h of massed and spaced training at varying intervals. Despite similar acquisition by all training protocols, 1 h of spacing produced the highest memory retention at 24 h, which lasted for 1 mo. The distinct kinetics of memory are strongly correlated with the reduction of floccular parallel fiber-Purkinje cell synapses but not with AMPA receptor (AMPAR) number and synapse size. After the spaced training, we observed 25%, 23%, and 12% reduction in AMPAR density, synapse size, and synapse number, respectively. Four hours after the spaced training, half of the synapses and Purkinje cell spines had been eliminated, whereas AMPAR density and synapse size were recovered in remaining synapses. Surprisingly, massed training also produced long-term memory and halving of synapses; however, this occurred slowly over days, and the memory lasted for only 1 wk. This distinct kinetics of structural plasticity may serve as a basis for unique temporal profiles in the formation and decay of memory with or without intervals. AU - Aziz, Wajeeha AU - Wang, Wen AU - Kesaf, Sebnem AU - Mohamed, Alsayed AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi ID - 1919 IS - 1 JF - PNAS TI - Distinct kinetics of synaptic structural plasticity, memory formation, and memory decay in massed and spaced learning VL - 111 ER - TY - JOUR AB - The development of the vertebrate brain requires an exquisite balance between proliferation and differentiation of neural progenitors. Notch signaling plays a pivotal role in regulating this balance, yet the interaction between signaling and receiving cells remains poorly understood. We have found that numerous nascent neurons and/or intermediate neurogenic progenitors expressing the ligand of Notch retain apical endfeet transiently at the ventricular lumen that form adherens junctions (AJs) with the endfeet of progenitors. Forced detachment of the apical endfeet of those differentiating cells by disrupting AJs resulted in precocious neurogenesis that was preceded by the downregulation of Notch signaling. Both Notch1 and its ligand Dll1 are distributed around AJs in the apical endfeet, and these proteins physically interact with ZO-1, a constituent of the AJ. Furthermore, live imaging of a fluorescently tagged Notch1 demonstrated its trafficking from the apical endfoot to the nucleus upon cleavage. Our results identified the apical endfoot as the central site of active Notch signaling to securely prohibit inappropriate differentiation of neural progenitors. AU - Hatakeyama, Jun AU - Wakamatsu, Yoshio AU - Nagafuchi, Akira AU - Kageyama, Ryoichiro AU - Shigemoto, Ryuichi AU - Shimamura, Kenji ID - 1933 IS - 8 JF - Development TI - Cadherin-based adhesions in the apical endfoot are required for active Notch signaling to control neurogenesis in vertebrates VL - 141 ER - TY - JOUR AB - Synaptic cell adhesion molecules are increasingly gaining attention for conferring specific properties to individual synapses. Netrin-G1 and netrin-G2 are trans-synaptic adhesion molecules that distribute on distinct axons, and their presence restricts the expression of their cognate receptors, NGL1 and NGL2, respectively, to specific subdendritic segments of target neurons. However, the neural circuits and functional roles of netrin-G isoform complexes remain unclear. Here, we use netrin-G-KO and NGL-KO mice to reveal that netrin-G1/NGL1 and netrin-G2/NGL2 interactions specify excitatory synapses in independent hippocampal pathways. In the hippocampal CA1 area, netrin-G1/NGL1 and netrin-G2/NGL2 were expressed in the temporoammonic and Schaffer collateral pathways, respectively. The lack of presynaptic netrin-Gs led to the dispersion of NGLs from postsynaptic membranes. In accord, netrin-G mutant synapses displayed opposing phenotypes in long-term and short-term plasticity through discrete biochemical pathways. The plasticity phenotypes in netrin-G-KOs were phenocopied in NGL-KOs, with a corresponding loss of netrin-Gs from presynaptic membranes. Our findings show that netrin-G/NGL interactions differentially control synaptic plasticity in distinct circuits via retrograde signaling mechanisms and explain how synaptic inputs are diversified to control neuronal activity. AU - Matsukawa, Hiroshi AU - Akiyoshi Nishimura, Sachiko AU - Zhang, Qi AU - Luján, Rafael AU - Yamaguchi, Kazuhiko AU - Goto, Hiromichi AU - Yaguchi, Kunio AU - Hashikawa, Tsutomu AU - Sano, Chie AU - Shigemoto, Ryuichi AU - Nakashiba, Toshiaki AU - Itohara, Shigeyoshi ID - 2018 IS - 47 JF - Journal of Neuroscience SN - 0270-6474 TI - Netrin-G/NGL complexes encode functional synaptic diversification VL - 34 ER - TY - JOUR AB - We examined the synaptic structure, quantity, and distribution of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-type glutamate receptors (AMPARs and NMDARs, respectively) in rat cochlear nuclei by a highly sensitive freeze-fracture replica labeling technique. Four excitatory synapses formed by two distinct inputs, auditory nerve (AN) and parallel fibers (PF), on different cell types were analyzed. These excitatory synapse types included AN synapses on bushy cells (AN-BC synapses) and fusiform cells (AN-FC synapses) and PF synapses on FC (PF-FC synapses) and cartwheel cell spines (PF-CwC synapses). Immunogold labeling revealed differences in synaptic structure as well as AMPAR and NMDAR number and/or density in both AN and PF synapses, indicating a target-dependent organization. The immunogold receptor labeling also identified differences in the synaptic organization of FCs based on AN or PF connections, indicating an input-dependent organization in FCs. Among the four excitatory synapse types, the AN-BC synapses were the smallest and had the most densely packed intramembrane particles (IMPs), whereas the PF-CwC synapses were the largest and had sparsely packed IMPs. All four synapse types showed positive correlations between the IMP-cluster area and the AMPAR number, indicating a common intrasynapse-type relationship for glutamatergic synapses. Immunogold particles for AMPARs were distributed over the entire area of individual AN synapses; PF synapses often showed synaptic areas devoid of labeling. The gold-labeling for NMDARs occurred in a mosaic fashion, with less positive correlations between the IMP-cluster area and the NMDAR number. Our observations reveal target- and input-dependent features in the structure, number, and organization of AMPARs and NMDARs in AN and PF synapses. AU - Rubio, Maía AU - Fukazawa, Yugo AU - Kamasawa, Naomi AU - Clarkson, Cheryl AU - Molnár, Elek AU - Shigemoto, Ryuichi ID - 2064 IS - 18 JF - Journal of Comparative Neurology TI - Target- and input-dependent organization of AMPA and NMDA receptors in synaptic connections of the cochlear nucleus VL - 522 ER - TY - JOUR AB - The brain demands high-energy supply and obstruction of blood flow causes rapid deterioration of the healthiness of brain cells. Two major events occur upon ischemia: acidosis and liberation of excess glutamate, which leads to excitotoxicity. However, cellular source of glutamate and its release mechanism upon ischemia remained unknown. Here we show a causal relationship between glial acidosis and neuronal excitotoxicity. As the major cation that flows through channelrhodopsin-2 (ChR2) is proton, this could be regarded as an optogenetic tool for instant intracellular acidification. Optical activation of ChR2 expressed in glial cells led to glial acidification and to release of glutamate. On the other hand, glial alkalization via optogenetic activation of a proton pump, archaerhodopsin (ArchT), led to cessation of glutamate release and to the relief of ischemic brain damage in vivo. Our results suggest that controlling glial pH may be an effective therapeutic strategy for intervention of ischemic brain damage. AU - Beppu, Kaoru AU - Sasaki, Takuya AU - Tanaka, Kenji AU - Yamanaka, Akihiro AU - Fukazawa, Yugo AU - Shigemoto, Ryuichi AU - Matsui, Ko ID - 2241 IS - 2 JF - Neuron SN - 08966273 TI - Optogenetic countering of glial acidosis suppresses glial glutamate release and ischemic brain damage VL - 81 ER - TY - JOUR AB - Despite the pivotal functions of the NMDA receptor (NMDAR) for neural circuit development and synaptic plasticity, the molecular mechanisms underlying the dynamics of NMDAR trafficking are poorly understood. The cell adhesion molecule neuroligin-1 (NL1) modifies NMDAR-dependent synaptic transmission and synaptic plasticity, but it is unclear whether NL1 controls synaptic accumulation or function of the receptors. Here, we provide evidence that NL1 regulates the abundance of NMDARs at postsynaptic sites. This function relies on extracellular, NL1 isoform-specific sequences that facilitate biochemical interactions between NL1 and the NMDAR GluN1 subunit. Our work uncovers NL1 isoform-specific cisinteractions with ionotropic glutamate receptors as a key mechanism for controlling synaptic properties. AU - Budreck, Elaine C AU - Kwon, Oh-Bin AU - Jung, Jung-Hoon AU - Baudouin, Stéphane J AU - Thommen, Albert AU - Kim, Hye-Sun AU - Fukazawa, Yugo AU - Harumi Harada AU - Tabuchi, Katsuhiko AU - Ryuichi Shigemoto AU - Scheiffele, Peter AU - Kim, Joung-Hun ID - 2478 IS - 2 JF - PNAS TI - Neuroligin-1 controls synaptic abundance of NMDA-type glutamate receptors through extracellular coupling VL - 110 ER - TY - JOUR AB - The group III metabotropic glutamate (mGlu) receptors mGlu7 and mGlu8 are receiving increased attention as potential novel therapeutic targets for anxiety disorders. The effects mediated by these receptors appear to result from a complex interplay of facilitatory and inhibitory actions at different brain sites in the anxiety/fear circuits. To better understand the effect of mGlu7 and mGlu8 receptors on extinction of contextual fear and their critical sites of action in the fear networks, we focused on the amygdala. Direct injection into the basolateral complex of the amygdala of the mGlu7 receptor agonist AMN082 facilitated extinction, whereas the mGlu8 receptor agonist (S)-3,4-DCPG sustained freezing during the extinction acquisition trial. We also determined at the ultrastructural level the synaptic distribution of these receptors in the basal nucleus (BA) and intercalated cell clusters (ITCs) of the amygdala. Both areas are thought to exert key roles in fear extinction. We demonstrate that mGlu7 and mGlu8 receptors are located in different presynaptic terminals forming both asymmetric and symmetric synapses, and that they preferentially target neurons expressing mGlu1α receptors mostly located around ITCs. In addition we show that mGlu7 and mGlu8 receptors were segregated to different inputs to a significant extent. In particular, mGlu7a receptors were primarily onto glutamatergic afferents arising from the BA or midline thalamic nuclei, but not the medial prefrontal cortex (mPFC), as revealed by combined anterograde tracing and pre-embedding electron microscopy. On the other hand, mGlu8a showed a more restricted distribution in the BA and appeared absent from thalamic, mPFC and intrinsic inputs. This segregation of mGlu7 and mGlu8 receptors in different neuronal pathways of the fear circuit might explain the distinct effects on fear extinction training observed with mGlu7 and mGlu8 receptor agonists. AU - Dobi, Alice AU - Sartori, Simone B AU - Busti, Daniela AU - Van Der Putten, Herman V AU - Singewald, Nicolas AU - Ryuichi Shigemoto AU - Ferraguti, Francesco ID - 2692 JF - Neuropharmacology TI - Neural substrates for the distinct effects of presynaptic group III metabotropic glutamate receptors on extinction of contextual fear conditioning in mice VL - 66 ER - TY - JOUR AB - P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Cav2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Cav2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Cav2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of 40∼nm. Calcium nanodomain created by the opening of Cav2.1 channels likely activates the two channels that limit the extent of depolarization. AU - Indriati, Dwi Wahyu AU - Kamasawa, Naomi AU - Matsui, Ko AU - Meredith, Andrea L AU - Watanabe, Masahiko AU - Ryuichi Shigemoto ID - 2691 IS - 8 JF - Journal of Neuroscience TI - Quantitative localization of Cav2.1 (P/Q-Type) voltage-dependent calcium channels in Purkinje cells: Somatodendritic gradient and distinct somatic coclustering with calcium-activated potassium channels VL - 33 ER - TY - JOUR AB - Establishing the spatiotemporal concentration profile of neurotransmitter following synaptic vesicular release is essential for our understanding of inter-neuronal communication. Such profile is a determinant of synaptic strength, short-term plasticity and inter-synaptic crosstalk. Synaptically released glutamate has been suggested to reach a few millimolar in concentration and last for <1 ms. The synaptic cleft is often conceived as a single concentration compartment, whereas a huge gradient likely exists. Modelling studies have attempted to describe this gradient, but two key parameters, the number of glutamate in a vesicle (NGlu) and its diffusion coefficient (DGlu) in the extracellular space, remained unresolved. To determine this profile, the rat calyx of Held synapse at postnatal day 12-16 was studied where diffusion of glutamate occurs two-dimensionally and where quantification of AMPA receptor distribution on individual postsynaptic specialization on medial nucleus of the trapezoid body principal cells is possible using SDS-digested freeze-fracture replica labelling. To assess the performance of these receptors as glutamate sensors, a kinetic model of the receptors was constructed from outside-out patch recordings. From here, we simulated synaptic responses and compared them with the EPSC recordings. Combinations of NGlu and DGlu with an optimum of 7000 and 0.3 μm2 ms-1 reproduced the data, suggesting slow diffusion. Further simulations showed that a single vesicle does not saturate the synaptic receptors, and that glutamate spillover does not affect the conductance amplitude at this synapse. Using the estimated profile, we also evaluated how the number of multiple vesicle releases at individual active zones affects the amplitude of postsynaptic signals. AU - Budisantoso, Timotheus AU - Harumi Harada AU - Kamasawa, Naomi AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Matsui, Ko ID - 2690 IS - 1 JF - Journal of Physiology TI - Evaluation of glutamate concentration transient in the synaptic cleft of the rat calyx of Held VL - 591 ER - TY - JOUR AB - Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gammafrequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABAB receptors (GABABRs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABABRs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity forGABABRs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABABR-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABABR activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABAB IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABABRs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABABRs may shift the balance between perisomatic and dendritic inhibition. AU - Booker, Sam A AU - Gross, Anna AU - Althof, Daniel AU - Ryuichi Shigemoto AU - Bettler, Bernhard AU - Frotscher, Michael AU - Hearing, Matthew C AU - Wickman, Kevin D AU - Watanabe, Masahiko AU - Kulik, Ákos AU - Vida, Imre ID - 2693 IS - 18 JF - Journal of Neuroscience TI - Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons VL - 33 ER - TY - JOUR AB - Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA ARγ2 77Ilox) in which the γ2 subunit of the GABA A receptor (GABA AR) was mutated to become zolpidem insensitive (γ2 77I) and used viral vectors to swap γ2 77I with wild-type, zolpidem-sensitive γ2 subunits (γ2 77F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA ARγ2 -/- mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ( AU1γ2 77F) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the AU1γ2 77F subunit (Cre-2A- AU1γ2 77F) into the neocortex of GABA ARγ2 77Ilox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the AU1γ2 77F subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2 77I and AU1γ2 77F GABA ARs in cortical pyramidal cells. This transgenic-viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations. AU - Sümegi, Máté AU - Fukazawa, Yugo AU - Matsui, Ko AU - Lörincz, Andrea AU - Eyre, Mark D AU - Nusser, Zoltán AU - Ryuichi Shigemoto ID - 2476 IS - 7 JF - Journal of Physiology TI - Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA A receptors in cortical pyramidal cells VL - 590 ER - TY - JOUR AB - Background: One of the best-characterized causative factors of Alzheimer's disease (AD) is the generation of amyloid-β peptide (Aβ). AD subjects are at high risk of epileptic seizures accompanied by aberrant neuronal excitability, which in itself enhances Aβ generation. However, the molecular linkage between epileptic seizures and Aβ generation in AD remains unclear. Results: X11 and X11-like (X11L) gene knockout mice suffered from epileptic seizures, along with a malfunction of hyperpolarization-activated cyclic nucleotide gated (HCN) channels. Genetic ablation of HCN1 in mice and HCN1 channel blockage in cultured Neuro2a (N2a) cells enhanced Aβ generation. Interestingly, HCN1 levels dramatically decreased in the temporal lobe of cynomolgus monkeys (Macaca fascicularis) during aging and were significantly diminished in the temporal lobe of sporadic AD patients. Conclusion: Because HCN1 associates with amyloid-β precursor protein (APP) and X11/X11L in the brain, genetic deficiency of X11/X11L may induce aberrant HCN1 distribution along with epilepsy. Moreover, the reduction in HCN1 levels in aged primates may contribute to augmented Aβ generation. Taken together, HCN1 is proposed to play an important role in the molecular linkage between epileptic seizures and Aβ generation, and in the aggravation of sporadic AD. AU - Saito, Yuhki AU - Inoue, Tsuyoshi AU - Zhu, Gang AU - Kimura, Naoki AU - Okada, Motohiro AU - Nishimura, Masaki AU - Murayama, Shigeo AU - Kaneko, Sunao AU - Ryuichi Shigemoto AU - Imoto, Keiji AU - Suzuki, Toshiharu ID - 2475 IS - 1 JF - Molecular Neurodegeneration TI - Hyperpolarization-activated cyclic nucleotide gated channels: A potential molecular link between epileptic seizures and Aβ generation in Alzheimer's disease VL - 7 ER - TY - JOUR AB - Dynamic activity of glia has repeatedly been demonstrated, but if such activity is independent from neuronal activity, glia would not have any role in the information processing in the brain or in the generation of animal behavior. Evidence for neurons communicating with glia is solid, but the signaling pathway leading back from glial-to-neuronal activity was often difficult to study. Here, we introduced a transgenic mouse line in which channelrhodopsin-2, a light-gated cation channel, was expressed in astrocytes. Selective photostimulation of these astrocytes in vivo triggered neuronal activation. Using slice preparations, we show that glial photostimulation leads to release of glutamate, which was sufficient to activate AMPA receptors on Purkinje cells and to induce long-term depression of parallel fiber-to-Purkinje cell synapses through activation of metabotropic glutamate receptors. In contrast to neuronal synaptic vesicular release, glial activation likely causes preferential activation of extrasynaptic receptors that appose glial membrane. Finally, we show that neuronal activation by glial stimulation can lead to perturbation of cerebellar modulated motor behavior. These findings demonstrate that glia can modulate the tone of neuronal activity and behavior. This animal model is expected to be a potentially powerful approach to study the role of glia in brain function. AU - Sasaki, Takuya AU - Beppu, Kaoru AU - Tanaka, Kenji F AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Matsui, Ko ID - 2477 IS - 50 JF - PNAS TI - Application of an optogenetic byway for perturbing neuronal activity via glial photostimulation VL - 109 ER - TY - JOUR AB - Interneurons are critical for neuronal circuit function, but how their dendritic morphologies and membrane properties influence information flow within neuronal circuits is largely unknown. We studied the spatiotemporal profile of synaptic integration and short-term plasticity in dendrites of mature cerebellar stellate cells by combining two-photon guided electrical stimulation, glutamate uncaging, electron microscopy, and modeling. Synaptic activation within thin (0.4 μm) dendrites produced somatic responses that became smaller and slower with increasing distance from the soma, sublinear subthreshold input-output relationships, and a somatodendritic gradient of short-term plasticity. Unlike most studies showing that neurons employ active dendritic mechanisms, we found that passive cable properties of thin dendrites determine the sublinear integration and plasticity gradient, which both result from large dendritic depolarizations that reduce synaptic driving force. These integrative properties allow stellate cells to act as spatiotemporal filters of synaptic input patterns, thereby biasing their output in favor of sparse presynaptic activity. Stellate cells are critical sources of inhibition in the cerebellum, but how their dendrites integrate excitatory synaptic inputs is unknown. Abrahamsson et al. show that thin dendrites and passive membrane properties of SCs promote sublinear synaptic summation and distance-dependent short-term plasticity. AU - Abrahamsson, Therese AU - Cathala, Laurence AU - Matsui, Ko AU - Ryuichi Shigemoto AU - DiGregorio, David A ID - 2474 IS - 6 JF - Neuron TI - Thin dendrites of cerebellar interneurons confer sublinear synaptic integration and a gradient of short-term plasticity VL - 73 ER - TY - JOUR AB - We investigated the temporal and spatial expression of SK2 in the developing mouse hippocampus using molecular and biochemical techniques, quantitative immunogold electron microscopy, and electrophysiology. The mRNA encoding SK2 was expressed in the developing and adult hippocampus. Western blotting and immunohistochemistry showed that SK2 protein increased with age. This was accompanied by a shift in subcellular localization. Early in development (P5), SK2 was predominantly localized to the endoplasmic reticulum in the pyramidal cell layer. But by P30 SK2 was almost exclusively expressed in the dendrites and spines. The level of SK2 at the postsynaptic density (PSD) also increased during development. In the adult, SK2 expression on the spine plasma membrane showed a proximal-to-distal gradient. Consistent with this redistribution and gradient of SK2, the selective SK channel blocker apamin increased evoked excitatory postsynaptic potentials (EPSPs) only in CA1 pyramidal neurons from mice older than P15. However, the effect of apamin on EPSPs was not different between synapses in proximal or distal stratum radiatum or stratum lacunosum-moleculare in adult. These results show a developmental increase and gradient in SK2-containing channel surface expression that underlie their influence on neurotransmission, and that may contribute to increased memory acquisition during early development. AU - Ballesteros-Merino, Carmen AU - Lin, Michael AU - Wu, Wendy W AU - Ferrándiz-Huertas, Clotilde AU - Cabañero, María José AU - Watanabe, Masahiko AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Maylie, James G AU - Adelman, John P AU - Luján, Rafael ID - 2515 IS - 6 JF - Hippocampus TI - Developmental profile of SK2 channel expression and function in CA1 neurons VL - 22 ER - TY - JOUR AB - Visual information must be relayed through the lateral geniculate nucleus before it reaches the visual cortex. However, not all spikes created in the retina lead to postsynaptic spikes and properties of the retinogeniculate synapse contribute to this filtering. To understand the mechanisms underlying this filtering process, we conducted electrophysiology to assess the properties of signal transmission in the Long-Evans rat. We also performed SDS-digested freeze-fracture replica labeling to quantify the receptor and transporter distribution, as well as EM reconstruction to describe the 3D structure. To analyze the impact of transmitter diffusion on the activity of the receptors, simulations were integrated. We identified that a large contributor to the filtering is the marked paired-pulse depression at this synapse, which was intensified by the morphological characteristics of the contacts. The broad presynaptic and postsynaptic contact area restricts transmitter diffusion two dimensionally. Additionally, the presence of multiple closely arranged release sites invites intersynaptic spillover, which causes desensitization of AMPA receptors. The presence of AMPA receptors that slowly recover from desensitization along with the high presynaptic release probability and multivesicular release at each synapse also contribute to the depression. These features contrast with many other synapses where spatiotemporal spread of transmitter is limited by rapid transmitter clearance allowing synapses to operate more independently. We propose that the micrometer-order structure can ultimately affect the visual information processing. AU - Budisantoso, Timotheus AU - Matsui, Ko AU - Kamasawa, Naomi AU - Fukazawa, Yugo AU - Ryuichi Shigemoto ID - 2514 IS - 7 JF - Journal of Neuroscience TI - Mechanisms underlying signal filtering at a multisynapse contact VL - 32 ER - TY - JOUR AB - R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Ca v2.3 in mice and rats at both light and electron microscopic levels. Ca v2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained <20 immunogold particles, with an average density of ~260 immunoparticles per μm 3 spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Ca v2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites. AU - Parajuli, Laxmi K AU - Nakajima, Chikako AU - Kulik, Ákos AU - Matsui, Ko AU - Schneider, Toni AU - Ryuichi Shigemoto AU - Fukazawa, Yugo ID - 2689 IS - 39 JF - Journal of Neuroscience TI - Quantitative regional and ultra structural localization of the Ca v2 3 subunit of R type calcium channel in mouse brain VL - 32 ER - TY - JOUR AB - Left-right asymmetry of human brain function has been known for a century, although much of molecular and cellular basis of brain laterality remains to be elusive. Recent studies suggest that hippocampal CA3-CA1 excitatory synapses are asymmetrically arranged, however, the functional implication of the asymmetrical circuitry has not been studied at the behavioral level. In order to address the left-right asymmetry of hippocampal function in behaving mice, we analyzed the performance of "split-brain" mice in the Barnes maze. The "split-brain" mice received ventral hippocampal commissure and corpus callosum transection in addition to deprivation of visual input from one eye. In such mice, the hippocampus in the side of visual deprivation receives sensory-driven input. Better spatial task performance was achieved by the mice which were forced to use the right hippocampus than those which were forced to use the left hippocampus. In two-choice spatial maze, forced usage of left hippocampus resulted in a comparable performance to the right counterpart, suggesting that both hippocampal hemispheres are capable of conducting spatial learning. Therefore, the results obtained from the Barnes maze suggest that the usage of the right hippocampus improves the accuracy of spatial memory. Performance of non-spatial yet hippocampus-dependent tasks (e.g. fear conditioning) was not influenced by the laterality of the hippocampus. AU - Shinohara, Yoshiaki AU - Hosoya, Aki AU - Yamasaki, Nobuyuki AU - Ahmed, Hassan AU - Hattori, Satoko AU - Eguchi, Megumi AU - Yamaguchi, Shun AU - Miyakawa, Tsuyoshi AU - Hirase, Hajime AU - Ryuichi Shigemoto ID - 2687 IS - 2 JF - Hippocampus TI - Right-hemispheric dominance of spatial memory in split-brain mice VL - 22 ER - TY - JOUR AB - To gain insights into structure-function relationship of excitatory synapses, we revisit our quantitative analysis of synaptic AMPAR by highly sensitive freeze-fracture replica labeling in eight different connections. All of these connections showed linear correlation between synapse size and AMPAR number indicating a common intra-synapse-type relationship in CNS synapses. On the contrary, inter-synapse-type relationship is unexpected indicating no correlation between averages of synapse size and AMPAR number. Interestingly, connections with large average synapse size and low AMPAR density showed high variability of AMPAR number and mosaic distribution within the postsynaptic membrane. We propose an idea that these connections may quickly exhibit synaptic plasticity by modifying AMPAR density/number whereas those with high AMPAR density change their efficacy by modifying synapse size. AU - Fukazawa, Yugo AU - Ryuichi Shigemoto ID - 2688 IS - 3 JF - Current Opinion in Neurobiology TI - Intra-synapse-type and inter-synapse-type relationships between synaptic size and AMPAR expression VL - 22 ER - TY - JOUR AB - Parkinson's disease is a common neurodegenerative disorder characterized by a profound motor disability that is traceable to the emergence of synchronous, rhythmic spiking in neurons of the external segment of the globus pallidus (GPe). The origins of this pathophysiology are poorly defined for the generation of pacemaking. After the induction of a parkinsonian state in mice, there was a progressive decline in autonomous GPe pacemaking, which normally serves to desynchronize activity. The loss was attributable to the downregulation of an ion channel that is essential in pacemaking, the hyperpolarization and cyclic nucleotide-gated (HCN) channel. Viral delivery of HCN2 subunits restored pacemaking and reduced burst spiking in GPe neurons. However, the motor disability induced by dopamine (DA) depletion was not reversed, suggesting that the loss of pacemaking was a consequence, rather than a cause, of key network pathophysiology, a conclusion that is consistent with the ability of L-type channel antagonists to attenuate silencing after DA depletion. AU - Chan, Savio AU - Glajch, Kelly E AU - Gertler, Tracy S AU - Guzmán, Jaime N AU - Mercer, Jeff N AU - Lewis, Alan S AU - Goldberg, Alan B AU - Tkatch, Tatiana AU - Ryuichi Shigemoto AU - Fleming, Sheila M AU - Chetkovich, Dane M AU - Osten, Pavel AU - Kita, Hitoshi AU - Surmeier, James D ID - 2511 IS - 1 JF - Nature Neuroscience TI - HCN channelopathy in external globus pallidus neurons in models of Parkinson s disease VL - 14 ER - TY - JOUR AB - GABAergic inhibition plays a central role in the control of pyramidal cell ensemble activities; thus, any signaling mechanism that regulates inhibition is able to fine-tune network patterns. Here, we provide evidence that the retrograde nitric oxide (NO)- cGMP cascade triggered by NMDA receptor (NMDAR) activation plays a role in the control of hippocampal GABAergic transmission in mice. GABAergic synapses express neuronal nitric oxide synthase (nNOS) postsynaptically and NO receptors (NO-sensitive guanylyl cyclase) in the presynaptic terminals. We hypothesized that-similar to glutamatergic synapses-the Ca 2+ transients required to activate nNOS were provided by NMDA receptor activation. Indeed, administration of 5 μm NMDA induced a robust nNOS-dependent cGMP production in GABAergic terminals, selectively in the CA1 and CA3c areas. Furthermore, using preembedding, postembedding, and SDS-digested freeze-fracture replica immunogold labeling, we provided quantitative immunocytochemical evidence that NMDAR subunits GluN1, GluN2A, and GluN2B were present in most somatic GABAergic synapses postsynaptically. These data indicate that NMDARs can modulate hippocampal GABAergic inhibition via NO- cGMP signaling in an activity-dependent manner and that this effect is subregion specific in the mouse hippocampus. AU - Szabadits, Eszter AU - Cserép, Csaba AU - Szonyi, András AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Watanabe, Masahiko AU - Itohara, Shigeyoshi AU - Freund, Tamás F AU - Nyíri, Gábor ID - 2512 IS - 16 JF - Journal of Neuroscience TI - NMDA receptors in hippocampal GABAergic synapses and their role in nitric oxide signaling VL - 31 ER - TY - JOUR AB - SK2-containing channels are expressed in the postsynaptic density (PSD) of dendritic spines on mouse hippocampal area CA1 pyramidal neurons and influence synaptic responses, plasticity and learning. The Sk2 gene (also known as Kcnn2) encodes two isoforms that differ only in the length of their N-terminal domains. SK2-long (SK2-L) and SK2-short (SK2-S) are coexpressed in CA1 pyramidal neurons and likely form heteromeric channels. In mice lacking SK2-L (SK2-S only mice), SK2-S-containing channels were expressed in the extrasynaptic membrane, but were excluded from the PSD. The SK channel contribution to excitatory postsynaptic potentials was absent in SK2-S only mice and was restored by SK2-L re-expression. Blocking SK channels increased the amount of long-term potentiation induced in area CA1 in slices from wild-type mice but had no effect in slices from SK2-S only mice. Furthermore, SK2-S only mice outperformed wild-type mice in the novel object recognition task. These results indicate that SK2-L directs synaptic SK2-containing channel expression and is important for normal synaptic signaling, plasticity and learning. AU - Allen, Duane H AU - Bond, Chris T AU - Luján, Rafael AU - Ballesteros-Merino, Carmen AU - Lin, Michael T AU - Wang, Kang AU - Klett, Nathan AU - Watanabe, Masahiko AU - Ryuichi Shigemoto AU - Stackman, Robert W AU - Maylie, James G AU - Adelman, John P ID - 2513 IS - 6 JF - Nature Neuroscience TI - The SK2-long isoform directs synaptic localization and function of SK2-containing channels VL - 14 ER - TY - JOUR AB - Epilepsy is a devastating and poorly understood disease. Mutations in a secreted neuronal protein, leucine-rich glioma inactivated 1 (LGI1), were reported in patients with an inherited form of human epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF). Here, we report an essential role of LGI1 as an antiepileptogenic ligand. We find that loss of LGI1 in mice (LGI1-/-) causes lethal epilepsy, which is specifically rescued by the neuronal expression of LGI1 transgene, but not LGI3. Moreover, heterozygous mice for the LGI1 mutation (LGI1+/-) show lowered seizure thresholds. Extracellularly secreted LGI1 links two epilepsy-related receptors, ADAM22 and ADAM23, in the brain and organizes a transsynaptic protein complex that includes presynaptic potassium channels and postsynaptic AMPA receptor scaffolds. A lack of LGI1 disrupts this synaptic protein connection and selectively reduces AMPA receptor-mediated synaptic transmission in the hippocampus. Thus, LGI1 may serve as a major determinant of brain excitation, and the LGI1 gene-targeted mouse provides a good model for human epilepsy. AU - Fukata, Yuko AU - Lovero, Kathryn L AU - Iwanaga, Tsuyoshi AU - Watanabe, Atsushi AU - Yokoi, Norihiko AU - Tabuchi, Katsuhiko AU - Ryuichi Shigemoto AU - Nicoll, Roger A AU - Fukata, Masaki ID - 2503 IS - 8 JF - PNAS TI - Disruption of LGI1-linked synaptic complex causes abnormal synaptic transmission and epilepsy VL - 107 ER - TY - JOUR AB - The activity patterns of subthalamic nucleus (STN) neurons are intimately linked to motor function and dysfunction and arise through the complex interaction of intrinsic properties and inhibitory and excitatory synaptic inputs. In many neurons, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play key roles in intrinsic excitability and synaptic integration both under normal conditions and in disease states. However, in STN neurons, which strongly express HCN channels, their roles remain relatively obscure. To address this deficit, complementary molecular and cellular electrophysiological, imaging, and computational approaches were applied to the rat STN. Molecular profiling demonstrated that individual STN neurons express mRNA encoding several HCN subunits, with HCN2 and 3 being the most abundant. Light and electron microscopic analysis showed that HCN2 subunits are strongly expressed and distributed throughout the somatodendritic plasma membrane. Voltage-, current-, and dynamic-clamp analysis, two-photon Ca 2+ imaging, and computational modeling revealed that HCN channels are activated by GABA A receptor-mediated inputs and thus limit synaptic hyperpolarization and deinactivation of low-voltage-activated Ca 2+ channels. Although HCN channels also limited the temporal summation of EPSPs, generated through two-photon uncaging of glutamate, this action was largely shunted by GABAergic inhibition that was necessary for HCN channel activation. Together the data demonstrate that HCN channels in STN neurons selectively counteract GABA A receptor-mediated inhibition arising from the globus pallidus and thus promote single-spike activity rather than rebound burst firing. AU - Atherton, Jeremy F AU - Kitano, Katsunori AU - Baufreton, Jérôme AU - Fan, Kai AU - Wokosin, David L AU - Tkatch, Tatiana AU - Ryuichi Shigemoto AU - Surmeier, James D AU - Bevan, Mark D ID - 2508 IS - 47 JF - Journal of Neuroscience TI - Selective participation of somatodendritic HCN channels in inhibitory but not excitatory synaptic integration in neurons of the subthalamic nucleus VL - 30 ER - TY - JOUR AB - Hippocampal CA1 pyramidal cells, which receive γ-aminobutyric acid (GABA)ergic input from at least 18 types of presynaptic neuron, express 14 subunits of the pentameric GABAA receptor. The relative contribution of any subunit to synaptic and extrasynaptic receptors influences the dynamics of GABA and drug actions. Synaptic receptors mediate phasic GABA-evoked conductance and extrasynaptic receptors contribute to a tonic conductance. We used freeze-fracture replica-immunogold labelling, a sensitive quantitative immunocytochemical method, to detect synaptic and extrasynaptic pools of the alpha1, alpha2 and beta3 subunits. Antibodies to the cytoplasmic loop of the subunits showed immunogold particles concentrated on distinct clusters of intramembrane particles (IMPs) on the cytoplasmic face of the plasma membrane on the somata, dendrites and axon initial segments, with an abrupt decrease in labelling at the edge of the IMP cluster. Neuroligin-2, a GABAergic synapse-specific adhesion molecule, co-labels all beta3 subunit-rich IMP clusters, therefore we considered them synapses. Double-labelling for two subunits showed that virtually all somatic synapses contain the alpha1, alpha2 and beta3 subunits. The extrasynaptic plasma membrane of the somata, dendrites and dendritic spines showed low-density immunolabelling. Synaptic labelling densities on somata for the alpha1, alpha2 and beta3 subunits were 78-132, 94 and 79 times higher than on the extrasynaptic membranes, respectively. As GABAergic synapses occupy 0.72% of the soma surface, the fraction of synaptic labelling was 33-48 (alpha1), 40 (alpha2) and 36 (beta3)% of the total somatic surface immunolabelling. Assuming similar antibody access to all receptors, about 60% of these subunits are in extrasynaptic receptors. AU - Kasugai, Yu AU - Swinny, Jerome D AU - Roberts, John D AU - Dalezios, Yannis AU - Fukazawa, Yugo AU - Sieghart, Werner C AU - Ryuichi Shigemoto AU - Somogyi, Péter ID - 2509 IS - 11 JF - European Journal of Neuroscience TI - Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling VL - 32 ER - TY - JOUR AB - Neurons in the laterocapsular division of the central nucleus of the amygdala (CeC), which is known as the "nociceptive amygdala," receive glutamatergic inputs from the parabrachial nucleus (PB) and the basolateral nucleus of amygdala (BLA), which convey nociceptive information from the dorsal horn of the spinal cord and polymodal information from the thalamus and cortex, respectively. Here, we examined the ultrastructural properties of PB- and BLA-CeC synapses identified with EGFP-expressing lentivirus in rats. In addition, the density of synaptic AMPA receptors (AMPARs) on CeC neurons was studied by using highly sensitive SDS-digested freeze-fracture replica labeling (SDS-FRL). Afferents from the PB made asymmetrical synapses mainly on dendritic shafts (88%), whereas those from the BLA were on dendritic spines (81%). PB-CeC synapses in dendritic shafts were significantly larger (median 0.072 μm 2) than BLA-CeC synapses in spines (median 0.058 μm 2; P = 0.02). The dendritic shafts that made synapses with PB fibers were also significantly larger than those that made synapses with BLA fibers, indicating that the PB fibers make synapses on more proximal parts of dendrites than the BLA fibers. SDS-FRL revealed that almost all excitatory postsynaptic sites have AMPARs in the CeC. The density of AMPAR-specific gold particles in individual synapses was significantly higher in spine synapses (median 510 particles/μm 2) than in shaft synapses (median 427 particles/μm 2; P = 0.01). These results suggest that distinct synaptic impacts from PB- and BLA-CeC pathways contribute to the integration of nociceptive and polymodal information in the CeC. AU - Dong, Yu-Lin AU - Fukazawa, Yugo AU - Wang, Wen AU - Kamasawa, Naomi AU - Ryuichi Shigemoto ID - 2510 IS - 23 JF - Journal of Comparative Neurology TI - Differential postsynaptic compartments in the laterocapsular division of the central nucleus of amygdala for afferents from the parabrachial nucleus and the basolateral nucleus in the rat VL - 518 ER - TY - JOUR AB - Subepithelial fibroblasts of the intestinal villi, which form a contractile cellular network beneath the epithelium, are in close contact with epithelial cells, nerve varicosities, capillaries, smooth muscles and immune cells, and secrete extracellular matrix molecules, growth factors and cytokines, etc. Cultured subepithelial fibroblasts of the rat duodenal villi display various receptors such as endothelins, ATP, substance-P and bradykinin, and release ATP in response to mechanical stimulation. In this study, the presence of functional NK1 receptors (NK1R) was pharmacologically confirmed in primary culture by Ca 2+ measurement, and the effects of substance-P were measured in an acute preparation of epithelium-free duodenal villi from 2- to 3-week-old rats using a two-photon laser microscope. Substance-P elicited an increase in the intracellular Ca 2+ concentration and contraction of the subepithelial fibroblasts in culture and the isolated villi. The localization of NK1R and substance-P in the villi was examined by light and electron microscopic immunohistochemistry. NK1R-like immunoreactivity was intensely localized on the plasma membrane of villous subepithelial fibroblasts in 10-day- to 4-week-old rats and mice and was decreased or absent in adulthood. The pericryptal fibroblasts of the small and large intestine were NK1R immuno-negative. These villous subepithelial fibroblasts form synapse-like structures with both substance-P-immunopositive and -immunonegative nerve varicosities. Here, we propose that the mutual interaction between villous subepithelial fibroblasts and afferent neurons via substance-P and ATP plays important roles in the maturation of the structure and function of the small intestine. AU - Furuya, Sonoko AU - Furuya, Kishio AU - Ryuichi Shigemoto AU - Sokabe, Masahiro ID - 2506 IS - 2 JF - Cell and Tissue Research TI - Localization of NK1 receptors and roles of substance-P in subepithelial fibroblasts of rat intestinal villi VL - 342 ER - TY - JOUR AB - T-type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T-type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane-associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3-fold higher than that of the interneurons. These results provide ultrastructural evidence for cell-type-specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells. AU - Parajuli, Laxmi K AU - Fukazawa, Yugo AU - Watanabe, Masahiko AU - Ryuichi Shigemoto ID - 2507 IS - 21 JF - Journal of Comparative Neurology TI - Subcellular distribution of α1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus VL - 518 ER - TY - JOUR AB - Cbln1, secreted from cerebellar granule cells, and the orphan glutamate receptor 52 (GluD2), expressed by Purkinje cells, are essential for synapse integrity between these neurons in adult mice. Nevertheless, no endogenous binding partners for these molecules have been identified. We found that Cblnl binds directly to the N-terminal domain of GluD2. GluD2 expression by postsynaptic cells, combined with exogenously applied Cbln1, was necessary and sufficient to induce new synapses in vitro and in the adult cerebellum in vivo. Further, beads coated with recombinant Cbln1 directly induced presynaptic differentiation and indirectly caused clustering of postsynaptic molecules via GluD2. These results indicate that the Cbln1-GluD2 complex is a unique synapse organizer that acts bidirectionally on both pre- and postsynaptic components. AU - Matsuda, Keiko AU - Miura, Eriko AU - Miyazaki, Taisuke AU - Kakegawa, Wataru AU - Emi, Kyoichi AU - Narumi, Sakae AU - Fukazawa, Yugo AU - Ito-lshida, Aya AU - Kondo, Tetsuro AU - Ryuichi Shigemoto AU - Watanabe, Masahiko AU - Yuzaki, Michisuke ID - 2505 IS - 5976 JF - Science TI - Cbln1 is a ligand for an orphan glutamate receptor δ2, a bidirectional synapse organizer VL - 328 ER - TY - JOUR AB - We present a method for immunolabeling of multiple species of membrane proteins with high spatial resolution. It allows differentiation of equally sized very small markers with different chemical compositions, which leads to high labeling efficiency and reduces steric hindrance of closely spaced immunolabeled biomolecules. Markers such as CdSe/ZnS semiconductor quantum dots and colloidal gold particles are distinguished by differential contrast in high-angle annular detector dark-field STEM mode or by EDX microanalysis of their elemental contents. This method was tested by observation of labeled AMPA- and NMDA-type glutamate receptors on sodium-dodecyl-sulfate-digested replica prepared from rat hippocampus. To improve particle visibility and detectability, the replica films were made exclusively with carbon to avoid the high background of conventional platinum/carbon replica. Extension of the method is suggested by detection of 1.4 nm nanogold particles and its potential application in the biological imaging research. AU - Loukanov, Alexandre R AU - Kamasawa, Naomi AU - Danev, Radostin S AU - Ryuichi Shigemoto AU - Nagayama, Kuniaki ID - 2504 IS - 4 JF - Ultramicroscopy TI - Immunolocalization of multiple membrane proteins on a carbon replica with STEM and EDX VL - 110 ER - TY - JOUR AB - Activation of G protein-gated inwardly-rectifying K+ (GIRK or Kir3) channels by metabotropic gamma-aminobutyric acid (B) (GABAB) receptors is an essential signalling pathway controlling neuronal excitability and synaptic transmission in the brain. To investigate the relationship between GIRK channel subunits and GABAB receptors in cerebellar Purkinje cells at post- and pre-synaptic sites, we used biochemical, functional and immunohistochemical techniques. Co-immunoprecipitation analysis demonstrated that GIRK subunits are co-assembled with GABAB receptors in the cerebellum. Immunoelectron microscopy showed that the subunit composition of GIRK channels in Purkinje cell spines is compartment-dependent. Thus, at extrasynaptic sites GIRK channels are formed by GIRK1/GIRK2/GIRK3, post-synaptic densities contain GIRK2/GIRK3 and dendritic shafts contain GIRK1/GIRK3. The post-synaptic association of GIRK subunits with GABAB receptors in Purkinje cells is supported by the subcellular regulation of the ion channel and the receptor in mutant mice. At pre-synaptic sites, GIRK channels localized to parallel fibre terminals are formed by GIRK1/GIRK2/GIRK3 and co-localize with GABAB receptors. Consistent with this morphological evidence we demonstrate their functional interaction at axon terminals in the cerebellum by showing that GIRK channels play a role in the inhibition of glutamate release by GABAB receptors. The association of GIRK channels and GABA B receptors with excitatory synapses at both post- and pre-synaptic sites indicates their intimate involvement in the modulation of glutamatergic neurotransmission in the cerebellum. AU - Fernández-Alacid, Laura AU - Aguado, Carolina AU - Ciruela, Francisco AU - Martín, Ricardo J AU - Colón, José AU - Cabañero, María José AU - Gassmann, Martin AU - Watanabe, Masahiko AU - Ryuichi Shigemoto AU - Wickman, Kevin D AU - Bettler, Bernhard AU - Sánchez-Prieto, José AU - Luján, Rafael ID - 2498 IS - 4 JF - Journal of Neurochemistry TI - Subcellular compartment-specific molecular diversity of pre- and post-synaptic GABAB-activated GIRK channels in Purkinje cells VL - 110 ER - TY - JOUR AB - To examine the intrasynaptic arrangement of postsynaptic receptors in relation to the functional role of the synapse,we quantitatively analyzed the two-dimensional distribution of AMPA and NMDA receptors (AMPARs and NMDARs, respectively) using SDS-digested freeze-fracture replica labeling (SDS-FRL) and assessed the implication of distribution differences on the postsynaptic responses by simulation. In the dorsal lateral geniculate nucleus, corticogeniculate (CG) synapses were twice as large as retinogeniculate (RG) synapses but expressed similar numbers of AMPARs. Two-dimensional views of replicas revealed that AMPARs form microclusters in both synapses to a similar extent, resulting in larger AMPAR-lacking areas in the CG synapses. Despite the broad difference in the AMPAR distribution within a synapse, our simulations based on the actual receptor distributions suggested that the AMPAR quantal response at individual RG synapses is only slightly larger in amplitude, less variable, and faster in kinetics than that at CG synapses having a similar number of the receptors. NMDARs at the CG synapses were expressed twice as many as those in the RG synapses. Electrophysiological recordings confirmed a larger contribution of NMDAR relative to AMPAR-mediated responses in CG synapses. We conclude that synapse size and the density and distribution of receptors have minor influences on quantal responses and that the number of receptors acts as a predominant postsynaptic determinant of the synaptic strength mediated by both the AMPARs and NMDARs. AU - Tarusawa, Etsuko AU - Matsui, Ko AU - Budisantoso, Timotheus AU - Molnár, Elek AU - Watanabe, Masahiko AU - Matsui, Minoru AU - Fukazawa, Yugo AU - Ryuichi Shigemoto ID - 2500 IS - 41 JF - Journal of Neuroscience TI - Input-specific intrasynaptic arrangements of ionotropic glutamate receptors and their impact on postsynaptic responses VL - 29 ER - TY - JOUR AB - G protein-coupled receptors (GPCRs) have critical functions in intercellular communication. Although a wide range of different receptors have been identified in the same cells, the mechanism by which signals are integrated remains elusive. The ability of GPCRs to form dimers or larger hetero-oligomers is thought to generate such signal integration. We examined the molecular mechanisms responsible for the GABAB receptor-mediated potentiation of the mGlu receptor signalling reported in Purkinje neurons. We showed that this effect does not require a physical interaction between both receptors. Instead, it is the result of a more general mechanism in which the βγ subunits produced by the Gi-coupled GABAB receptor enhance the mGlu-mediated Gq response. Most importantly, this mechanism could be generally applied to other pairs of Gi- and Gq-coupled receptors and the signal integration varied depending on the time delay between activation of each receptor. Such a mechanism helps explain specific properties of cells expressing two different Gi- and Gq-coupled receptors activated by a single transmitter, or properties of GPCRs naturally coupled to both types of the G protein. AU - Rives, Marie L AU - Vol, Claire AU - Fukazawa, Yugo AU - Tinel, Norbert AU - Trinquet, Eric AU - Ayoub, Mohammed A AU - Ryuichi Shigemoto AU - Pin, Jean-Philippe AU - Prezèau, Laurent ID - 2499 IS - 15 JF - EMBO Journal TI - Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration VL - 28 ER - TY - JOUR AB - In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state. AU - Fukuda, Yoshiyuki AU - Fukazawa, Yugo AU - Danev, Radostin S AU - Ryuichi Shigemoto AU - Nagayama, Kuniaki ID - 2502 IS - 3 JF - Journal of Structural Biology TI - Tuning of the Zernike phase-plate for visualization of detailed ultrastructure in complex biological specimens VL - 168 ER - TY - JOUR AB - The brain-specific immediate early gene Arc/Arg3.1 is induced in response to a variety of stimuli, including sensory and behavior-linked neural activity. Here we report the generation of transgenic mice, termed TgArc/Arg3.1-d4EGFP, expressing a 4-h half-life form of enhanced green fluorescent protein (d4EGFP) under the control of the Arc/Arg3.1 promoter. We show that d4EGFP-mediated fluorescence faithfully reports Arc/Arg3.1 induction in response to physiological, pathological and pharmacological stimuli, and that this fluorescence permits electrical recording from activated neurons in the live mouse. Moreover, the fluorescent Arc/Arg3.1 indicator revealed activity changes in circumscribed brain areas in distinct modes of stress and in a mouse model of Alzheimer's disease. These findings identify the TgArc/Arg3.1-d4EGFP mouse as a versatile tool to monitor Arc/Arg3.1 induction in neural circuits, both in vitro and in vivo. AU - Grinevich, Valery V AU - Kolleker, Alexander AU - Eliava, Marina I AU - Takada, Naoki AU - Takuma, Hiroshi AU - Fukazawa, Yugo AU - Ryuichi Shigemoto AU - Kuhl, Dietmar AU - Waters, Jack AU - Seeburg, Peter H AU - Osten, Pavel ID - 2501 IS - 1 JF - Journal of Neuroscience Methods TI - Fluorescent Arc/Arg3.1 indicator mice: A versatile tool to study brain activity changes in vitro and in vivo VL - 184 ER - TY - JOUR AB - Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells. AU - Walter Kaufmann AU - Ferraguti, Francesco AU - Fukazawa, Yugo AU - Kasugai, Yu AU - Ryuichi Shigemoto AU - Laake, Petter AU - Sexton, Joseph A AU - Ruth, Peter AU - Wietzorrek, Georg AU - Knaus, Hans G AU - Storm, Johan F AU - Ottersen, Ole P ID - 2684 IS - 2 JF - Journal of Comparative Neurology TI - Large-conductance calcium-activated potassium channels in Purkinje cell plasma membranes are clustered at sites of hypolemmal microdomains VL - 515 ER - TY - JOUR AB - GABA B receptor subtypes are based on the subunit isoforms GABA B1a and GABA B1b, which associate with GABA B2 subunits to form pharmacologically indistinguishable GABA B(1a,2) and GABA B(1b,2) receptors. Studies with mice selectively expressing GABA B1a or GABA B1b subunits revealed that GABA B(1a,2) receptors are more abundant than GABA B(1b,2) receptors at glutamatergic terminals. Accordingly, it was found that GABA B(1a,2) receptors are more efficient than GABA B(1b,2) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA B(1a,2) and GABA B(1b,2) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA B1a than GABA B1b protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations ≥1 μM, both GABA B(1a,2) and GABA B(1b,2) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA B(1a,2) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA B(1a,2) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA B(1a,2) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA B receptors. Our data demonstrate that the difference in GABA B1a and GABA B1b protein levels at MF terminals is sufficient to produce a strictly GABA B1a-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA B1a and GABA B1b proteins is of regulatory relevance. AU - Guetg, Nicole AU - Seddik, Riad AU - Vigot, Réjan AU - Tureček, Rostislav AU - Gassmann, Martin AU - Vogt, Kaspar E AU - Bräuner-Osborne, Hans AU - Ryuichi Shigemoto AU - Kretz, Oliver AU - Frotscher, Michael AU - Kulik, Ákos AU - Bettler, Bernhard ID - 2680 IS - 5 JF - Journal of Neuroscience TI - The GABA B1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses VL - 29 ER - TY - JOUR AB - GABAb receptor (GABAbR)-mediated suppression of glutamate release is critical for limiting glutamatergic transmission across the central nervous system (CNS). Here we show that, upon tetanic stimulation of afferents to lateral amygdala, presynaptic GABAbR-mediated inhibition only occurs in glutamatergic inputs to principle neurons (PNs), not to interneurons (INs), despite the presence of GABAbR in terminals to both types of neurons. The selectivity is caused by differential local GABA accumulation; it requires GABA reuptake and parallels distinct spatial distributions of presynaptic GABAbR in terminals to PNs and INs. Moreover, GABAbR-mediated suppression of theta-burst-induced long-term potentiation (LTP) occurs only in the inputs to PNs, not to INs. Thus, target-cell-specific control of glutamate release by presynaptic GABAbR orchestrates the inhibitory dominance inside amygdala and might contribute to prevention of nonadaptive defensive behaviors. AU - Pan, Bingxing AU - Dong, Yu-Lin AU - Ito, Wataru AU - Yanagawa, Yuchio AU - Ryuichi Shigemoto AU - Morozov, Alexei A ID - 2683 IS - 6 JF - Neuron TI - Selective gating of glutamatergic inputs to excitatory neurons of amygdala by presynaptic GABAb receptor VL - 61 ER - TY - JOUR AB - The living cell imaging using a two-photon microscope using gold nanoplates and nanoparticle aggregates was demonstrated. The dimensions of the nanoplates were determined through scanning electron microscopy (SEM) and atomic force microscopy. The height of a 100 nm base-length nanotriangle was around 10 nm, while the height of 300 nm base-length nanotriangle was around 12 nm. A spectrophotometer was also used to determine the extinction spectra of gold nanoparticle colloids. Two-photon-induced photoluminescence (TPIPL) under far-field excitation was tested for gold nanoplates on a glass substrate using two-photon laser scanning microscopy (TPLSM). It was observed that living-cell microscopic imaging can be carried out with TPIPL from gold nanoplates and aggregated nanosphere. This method provided a platform for developing tools for biological and biomedical studies. AU - Jiang, Yuqiang AU - Horimoto, Noriko N AU - Imura, Kohei AU - Matsui, Ko AU - Ryuichi Shigemoto ID - 2682 IS - 22 JF - Advanced Materials TI - Bioimaging with two-photon-induced luminescence from triangular nanoplates and nanoparticle aggregates of gold VL - 21 ER - TY - JOUR AB - Conduction velocity (CV) of myelinated axons has been shown to be regulated by oligodendrocytes even after myelination has been completed. However, how myelinating oligodendrocytes regulate CV, and what the significance of this regulation is for normal brain function remain unknown. To address these questions, we analyzed a transgenic mouse line harboring extra copies of the myelin proteolipid protein 1 (plp1) gene (plp1tg/- mice) at 2 months of age. At this stage, the plp1tg/- mice have an unaffected myelin structure with a normally appearing ion channel distribution, but the CV in all axonal tracts tested in the CNS is greatly reduced. We also found decreased axonal diameters and slightly abnormal paranodal structures, both of which can be a cause for the reduced CV. Interestingly the plp1tg/- mice showed altered anxiety-like behaviors, reduced prepulse inhibitions, spatial learning deficits and working memory deficit, all of which are schizophrenia-related behaviors. Our results implicate that abnormalities in the neuron-glia interactions at the paranodal junctions can result in reduced CV in the CNS, which then induces behavioral abnormalities related to schizophrenia. AU - Tanaka, Hisataka AU - Ma, Jianmei AU - Tanaka, Kenji F AU - Takao, Keizo AU - Komada, Munekazu AU - Tanda, Koichi AU - Suzuki, Ayaka AU - Ishibashi, Tomoko AU - Baba, Hiroko AU - Isa, Tadashi AU - Ryuichi Shigemoto AU - Ono, Katsuhiko AU - Miyakawa, Tsuyoshi AU - Ikenaka, Kazuhiro ID - 2685 IS - 26 JF - Journal of Neuroscience TI - Mice with altered myelin proteolipid protein gene expression display cognitive deficits accompanied by abnormal neuron-glia interactions and decreased conduction velocities VL - 29 ER - TY - JOUR AB - Channelrhodopsin-2 (ChR2), one of the archea-type rhodopsins from green algae, is a potentially useful optogenetic tool for restoring vision in patients with photoreceptor degeneration, such as retinitis pigmentosa. If the ChR2 gene is transferred to retinal ganglion cells (RGCs), which send visual information to the brain, the RGCs may be repurposed to act as photoreceptors. In this study, by using a transgenic rat expressing ChR2 specifically in the RGCs under the regulation of a Thy-1.2 promoter, we tested the possibility that direct photoactivation of RGCs could restore effective vision. Although the contrast sensitivities of the optomotor responses of transgenic rats were similar to those observed in the wild-type rats, they were enhanced for visual stimuli of low-spatial frequency after the degeneration of native photoreceptors. This result suggests that the visual signals derived from the ChR2-expressing RGCs were reinterpreted by the brain to form behavior-related vision. AU - Tomita, Hiroshi AU - Sugano, Eriko AU - Fukazawa, Yugo AU - Isago, Hitomi AU - Sugiyama, Yuka AU - Hiroi, Teru AU - Ishizuka, Toru AU - Mushiake, Hajime AU - Kato, Megumi AU - Hirabayashi, Masumi AU - Ryuichi Shigemoto AU - Yawo, Hiromu AU - Tamai, Makoto ID - 2686 IS - 11 JF - PLoS One TI - Visual properties of transgenic rats harboring the channelrhodopsin-2 gene regulated by the thy-1.2 promoter VL - 4 ER - TY - JOUR AB - Cyclic nucleotide phosphodiesterase 10A (PDE10A) is a member of phosphodiesterase families that degrade cAMP and/or cGMP in distinct intracellular sites. PDE10A has a dual activity on hydrolysis of both cAMP and cGMP, and is prominently expressed in the striatum and the testis. Previous studies suggested that PDE10A is involved in regulation of locomotor activity and potentially related to psychosis, but concrete physiological roles of PDE10A remains elusive yet. In this study, we genetically inactivated PDE10A2, a prominent isoform of PDE10A in the brain, in mice, and demonstrate that PDE10A2 deficiency results in increased social interaction without any major influence on different other behaviors, along with increased levels of striatal cAMP. We also demonstrate that PDE10A2 is selectively distributed in medium spiny neurons, but not interneurons, of the striatal complex. Thus, our results establish a physiological role for PDE10A2 in regulating cAMP pathway and social interaction, and suggest that cAMP signaling cascade in striatal medium spiny neurons might be involved in regulating social interaction behavior in mice. AU - Sano, Hiromi AU - Nagai, Yumiko AU - Miyakawa, Tsuyoshi AU - Ryuichi Shigemoto AU - Yokoi, Mineto ID - 2497 IS - 2 JF - Journal of Neurochemistry TI - Increased social interaction in mice deficient of the striatal medium spiny neuron-specific phosphodiesterase 10A2 VL - 105 ER - TY - JOUR AB - Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that generate Ih currents are widely distributed in the brain and have been shown to contribute to various neuronal functions. In the present study, we investigated the functions of Ih in the motion-sensitive projection neurons [wide field vertical (WFV) cells] of the superior colliculus, a pivotal visual center for detection of and orientating to salient objects. Combination of whole cell recordings and immunohistochemical investigations suggested that HCN1 channels dominantly contribute to the Ih in WFV cells among HCN isoforms expressed in the superficial superior colliculus and mainly located on their expansive dendritic trees. We found that blocking Ih suppressed the initiation of short- and fixed-latency dendritic spike responses and led instead to long- and fluctuating-latency somatic spike responses to optic fiber stimulations. These results suggest that the dendritic Ih facilitates the dendritic initiation and/or propagation of action potentials and ensures that WFV cells generate spike responses to distal synaptic inputs in a sensitive and robustly time-locked manner, probably by acting as continuous depolarizing drive and fixing dendritic membrane potentials close to the spike threshold. These functions are different from known functions of dendritic Ih revealed in hippocampal and neocortical pyramidal cells, where they spatiotemporally limit the propagations of synaptic inputs along the apical dendrites by reducing dendritic membrane resistance. Thus we have revealed new functional aspects of Ih, and these dendritic properties are likely critical for visual motion processing in these neurons. AU - Endo, Toshiaki AU - Tarusawa, Etsuko AU - Notomi, Takuya AU - Kaneda, Katsuyuki AU - Hirabayashi, Masumi AU - Ryuichi Shigemoto AU - Isa, Tadashi ID - 2675 IS - 5 JF - Journal of Neurophysiology TI - Dendritic Ih ensures high-fidelity dendritic spike responses of motion-sensitive neurons in rat superior colliculus VL - 99 ER - TY - JOUR AB - Left-right (L-R) asymmetry is a fundamental feature of higher-order neural function. However, the molecular basis of brain asymmetry remains unclear. We recently reported L-R asymmetry of hippocampal circuitry caused by differential allocation of N-methyl-O-aspartate receptor (NMDAR) subunit GluRε2 (NR2B) in hippocambal synapses. Using electrophysiology and immunocytochemistry, here we analyzed the hippocampal circuitry of the inversus viscerum (iv) mouse that has a randomized laterality of internal organs. The iv mouse hippocampus lacks L-R asymmetry, it exhibits right isomerism in the synaptic distribution of the ε2 subunit, irrespective of the laterality of visceral organs. This independent right isomerism of the hippocampus is the first evidence that a distinct mechanism downstream of the iv mutation generates brain asymmetry. AU - Kawakami, Ryosuke AU - Dobi, Alice AU - Ryuichi Shigemoto AU - Ito, Isao ID - 2676 IS - 4 JF - PLoS One TI - Right isomerism of the brain in inversus viscerum mutant mice VL - 3 ER - TY - GEN AU - Fukazawa, Yugo AU - Tarusawa, Etsuko AU - Matsui, Ko AU - Ryuichi Shigemoto ID - 2674 IS - 4 Suppl T2 - Tanpakushitsu kakusan koso Protein nucleic acid enzyme TI - Ultrastructural insights of postsynaptic glutamate receptor organization VL - 53 ER - TY - JOUR AB - Mammalian retinas contain abundant neuronal gap junctions, particularly in the inner plexiform layer (IPL), where the two principal neuronal connexin proteins are Cx36 and Cx45. Currently undetermined are coupling relationships between these connexins and whether both are expressed together or separately in a neuronal subtype-specific manner. Although Cx45-expressing neurons strongly couple with Cx36-expressing neurons, possibly via heterotypic gap junctions, Cx45 and Cx36 failed to form functional heterotypic channels in vitro. We now show that Cx36 and Cx45 coexpressed in HeLa cells were colocalized in immunofluorescent puncta between contacting cells, demonstrating targeting/scaffolding competence for both connexins in vitro. However, Cx36 and Cx45 expressed separately did not form immunofluorescent puncta containing both connexins, supporting lack of heterotypic coupling competence. In IPL, 87% of Cx45-immunofluorescent puncta were colocalized with Cx36, supporting either widespread heterotypic coupling or bihomotypic coupling. Ultrastructurally, Cx45 was detected in 9% of IPL gap junction hemiplaques, 90-100% of which also contained Cx36, demonstrating connexin coexpression and cotargeting in virtually all IPL neurons that express Cx45. Moreover, double replicas revealed both connexins in separate domains mirrored on both sides of matched hemiplaques. With previous evidence that Cx36 interacts with PDZ1 domain of zonula occludens-1 (ZO-1), we show that Cx45 interacts with PDZ2 domain of ZO-1, and that Cx36, Cx45, and ZO-1 coimmunoprecipitate, suggesting that ZO-1 provides for coscaffolding of Cx45 with Cx36. These data document that in Cx45-expressing neurons of IPL, Cx45 is almost always accompanied by Cx36, forming "bihomotypic" gap junctions, with Cx45 structurally coupling to Cx45 and Cx36 coupling to Cx36. AU - Li, Xinbo AU - Kamasawa, Naomi AU - Ciolofan, Cristina AU - Olson, Carl O AU - Lu, Shijun AU - Davidson, Kimberly G AU - Yasumura, Thomas AU - Ryuichi Shigemoto AU - Rash, John E AU - Nagy, James I ID - 2678 IS - 39 JF - Journal of Neuroscience TI - Connexin45-containing neuronal gap junctions in rodent retina also contain connexin36 in both apposing hemiplaques, forming bihomotypic gap junctions, with scaffolding contributed by zonula occludens-1 VL - 28 ER - TY - JOUR AB - The medial septum (MS) is an indispensable component of the subcortical network which synchronizes the hippocampus at theta frequency during specific stages of information processing. GABAergic neurons exhibiting highly regular firing coupled to the hippocampal theta rhythm are thought to form the core of the MS rhythm-generating network. In recent studies the hyperpolarization-activated, cyclic nucleotide-gated non-selective cation (HCN) channel was shown to participate in theta synchronization of the medial septum. Here, we tested the hypothesis that HCN channel expression correlates with theta modulated firing behaviour of MS neurons by a combined anatomical and electrophysiological approach. HCN-expressing neurons represented a subpopulation of GABAergic cells in the MS partly overlapping with parvalbumin (PV)-containing neurons. Rhythmic firing in the theta frequency range was characteristic of all HCN-expressing neurons. In contrast, only a minority of HCN-negative cells displayed theta related activity. All HCN cells had tight phase coupling to hippocampal theta waves. As a group, PV-expressing HCN neurons had a marked bimodal phase distribution, whereas PV-immunonegative HCN neurons did not show group-level phase preference despite significant individual phase coupling. Microiontophoretic blockade of HCN channels resulted in the reduction of discharge frequency, but theta rhythmic firing was perturbed only in a few cases. Our data imply that HCN-expressing GABAergic neurons provide rhythmic drive in all phases of the hippocampal theta activity. In most MS theta cells rhythm genesis is apparently determined by interactions at the level of the network rather than by the pacemaking property of HCN channels alone. AU - Varga, Viktor AU - Hangya, Balázs AU - Kránitz, Kinga AU - Ludányi, Anikó AU - Zemankovics, Rita AU - Katona, István AU - Ryuichi Shigemoto AU - Freund, Tamás F AU - Borhegyi, Zsolt ID - 2677 IS - 16 JF - Journal of Physiology TI - The presence of pacemaker HCN channels identifies theta rhythmic GABAergic neurons in the medial septum VL - 586 ER - TY - JOUR AB - Ionotropic glutamate receptors play important roles in spinal processing of nociceptive sensory signals and induction of central sensitization in chronic pain. Here we applied highly sensitive freeze-fracture replica labeling to laminae I-II of the spinal dorsal horn of rats and investigated the numbers, densities, and colocalization of AMPA- and NMDA-type glutamate receptors at individual postsynaptic membrane specializations with a high resolution. All glutamatergic postsynaptic membranes in laminae I-II expressed AMPA receptors, and most of them (96%) were also immunoreactive for the NR1 subunit of NMDA receptors. The numbers of gold particles for AMPA and NMDA receptors at individual postsynaptic membranes showed a linear correlation with the size of postsynaptic membrane specializations and varied in the range of 8-214 and 5-232 with median values of 37 and 28, whereas their densities varied in the range of 325-3365/μm 2 and 102-2263/μm 2 with median values of 1115/μm 2 and 777/μm 2, respectively. Virtually all (99%) glutamatergic postsynaptic membranes expressed GluR2, and most of them (87%) were also immunoreactive for GluR1. The numbers of gold particles for pan-AMPA, NR1, and GluR2 subunits showed a linear correlation with the size of postsynaptic surface areas. Concerning GluR1, there may be two populations of synapses with high and low GluR1 densities. In synapses larger than 0.1 μm 2, GluR1 subunits were recovered in very low numbers. Differential expression of GluR1 and GluR2 subunits suggests regulation of AMPA receptor subunit composition by presynaptic mechanism. AU - Antal, Miklós AU - Fukazawa, Yugo AU - Eördögh, Mária AU - Muszil, Dóra AU - Molnár, Elek AU - Itakura, Makoto AU - Takahashi, Masami AU - Ryuichi Shigemoto ID - 2679 IS - 39 JF - Journal of Neuroscience TI - Numbers, densities, and colocalization of AMPA- and NMDA-type glutamate receptors at individual synapses in the superficial spinal dorsal horn of rats VL - 28 ER - TY - JOUR AB - Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits. AU - Shinohara, Yoshiaki AU - Hirase, Hajime AU - Watanabe, Masahiko AU - Itakura, Makoto AU - Takahashi, Masami AU - Ryuichi Shigemoto ID - 2681 IS - 49 JF - PNAS TI - Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors VL - 105 ER - TY - JOUR AB - Metabotropic glutamate receptor 4 (mGluR4) is localized mainly to presynaptic membranes in the brain. Rat neostriatum has been reported to contain two types of mGluR4-immunoreactive axon varicosities: small, weakly immunoreactive varicosities that were distributed randomly (type 1) and large, intensely immunoreactive ones that were often aligned linearly (type 2). In the present study, most type 1 terminals formed asymmetric synapses on dendritic spines, whereas type 2 terminals made symmetric synapses on dendritic shafts, showing immunoreactivity for GABAergic markers. After depletion of neostriatal neurons, type 2 but not type 1 varicosities were largely decreased in the damaged region. When medium-sized spiny neurons (MSNs) were labeled with Sindbis virus expressing membrane-targeted green fluorescent protein, mGluR4 immunoreactivity was observed on some varicosities of their axon collaterals in immunofluorescence and immunoelectron microscopies. Furthermore, type 2 varicosities were often positive for substance P but mostly negative for striatal interneuron markers and preproenkephalin. Thus, striatonigral/striato- entopeduncular MSNs are likely to be the largest source of type 2 mGluR4-immunopositive axon terminals in the neostriatum. Next, in the double-immunofluorescence study, almost all choline acetyltransferase (ChAT)-immunopositive and 41% of NK1 receptor-positive dendrites were heavily associated with type 2 mGluR4-immunoreactive varicosities. Neuronal nitric oxide synthase (nNOS)-positive dendrites, in contrast, seemed associated with only a few type 2 varicosities. Conversely, almost all type 2 varicosities were closely apposed to NK1 receptor-positive dendrites that were known to be derived from cholinergic and nNOS-producing interneurons. These findings indicate that the mGluR4-positive terminals of MSN axon collaterals selectively form synapses with neostriatal cholinergic interneurons. AU - Kuramoto, Eriko AU - Fujiyama, Fumino AU - Unzai, Tomo AU - Nakamura, Kouichi AU - Hioki, Hiroyuki AU - Furuta, Takahiro AU - Ryuichi Shigemoto AU - Ferraguti, Francesco AU - Kaneko, Takeshi ID - 2495 IS - 5 JF - Journal of Comparative Neurology TI - Metabotropic glutamate receptor 4-immunopositive terminals of medium-sized spiny neurons selectively form synapses with cholinergic interneurons in the rat neostriatum VL - 500 ER - TY - JOUR AB - Glutamate is one candidate for the neurotransmitters and/or neuromodulators involved in taste signaling in taste buds. Group II metabotropic glutamate receptors (mGluRs: mGluR2 and mGluR3) are known to function as presynaptic receptors that regulate the release of glutamate and/or other neurotransmitters in the central nervous system. Group II mGluRs are negatively linked to adenylyl cyclase through Gαi subunits and thereby reduce the turnover of cAMP. In rat taste tissues, a subset of adenylyl-cyclase-8-expressing taste cells coexpress the Gαi subunits gustducin and Gαi2. However, the expression patterns of group II mGluRs in rat taste tissues have not yet been elucidated. We have therefore examined the expression patterns of mGluR2, mGluR3, and gustducin in rat gustatory tissues. Reverse transcription/polymerase chain reaction assays have revealed that mGluR2 and mGluR3 mRNAs are expressed in the circumvallate papillae. In situ hybridization analyses have detected positive signals for mGluR2 and mGluR3 mRNAs only in the circumvallate taste buds. Among the fungiform, foliate, and circumvallate papillae, an antibody against mGluR2/3 labels a subset of taste bud cells and nerve fibers immediately beneath the taste lingual epithelium. Double-labeling experiments have demonstrated that mGluR2/3-positive cells coexpress gustducin. These results indicate that mGluR2 and mGluR3 are coupled to Gαi subunits and play roles in glutamate-mediated signaling in taste transductions. AU - Toyono, Takashi AU - Kataoka, Shinji AU - Seta, Yuji AU - Ryuichi Shigemoto AU - Toyoshima, Kuniaki ID - 2496 IS - 1 JF - Cell and Tissue Research TI - Expression of group II metabotropic glutamate receptors in rat gustatory papillae VL - 328 ER - TY - JOUR AB - Substance P (SP) is known to be a peptide that facilitates epileptic activity of principal cells in the hippocampus. Paradoxically, in other models, it was found to be protective against seizures by activating substance P receptor (SPR)-expressing interneurons. Thus, these cells appear to play an important role in the generation and regulation of epileptic seizures. The number, distribution, morphological features and input characteristics of SPR-immunoreactive cells were analyzed in surgically removed hippocampi of 28 temporal lobe epileptic patients and eight control hippocampi in order to examine their changes in epileptic tissues. SPR is expressed in a subset of inhibitory cells in the control human hippocampus, they are multipolar interneurons with smooth dendrites, present in all hippocampal subfields. This cell population is considerably different from SPR-positive cells of the rat hippocampus. The CA1 (cornu Ammonis subfield 1) region was chosen for the detailed morphological analysis of the SPR-immunoreactive cells because of its extreme vulnerability in epilepsy. The presence of various neurochemical markers identifies functionally distinct interneuron types, such as those responsible for perisomatic, dendritic or interneuron-selective inhibition. We found considerable colocalization of SPR with calbindin but not with parvalbumin, calretinin, cholecystokinin and somatostatin, therefore we suppose that SPR-positive cells participate mainly in dendritic inhibition. In the non-sclerotic CA1 region they are mainly preserved, whereas their number is decreased in the sclerotic cases. In the epileptic samples their morphology is considerably altered, they possessed more dendritic branches, which often became beaded. Analyses of synaptic coverage revealed that the ratio of symmetric synaptic input of SPR-immunoreactive cells has increased in epileptic samples. Our results suggest that SPR-positive cells are preserved while principal cells are present in the CA1 region, but show reactive changes in epilepsy including intense branching and growth of their dendritic arborization. AU - Tóth, Kinga AU - Wittner, Lucia AU - Urbán, Z AU - Doyle, Werner K AU - Buzsáki, György AU - Ryuichi Shigemoto AU - Freund, Tamás F AU - Maglóczky, Zsófia ID - 2665 IS - 2 JF - Neuroscience TI - Morphology and synaptic input of substance P receptor-immunoreactive interneurons in control and epileptic human hippocampus VL - 144 ER - TY - JOUR AB - GABAB receptors (GABABRs) are involved in early events during neuronal development. The presence of GABABRs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABABR proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABAB1abcdf and GABAB2 subunits of the GABABR. Using semiquantitative PCR analysis with GABABR isoform-selective primers we found that the expression level of GABAB1abd was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABAB2 isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABAB1 and GABAB2 subunits are under independent control during oligodendroglial development. Activation of GABABRs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABABR activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABABRs in oligodendrocyte development. The presence of GABABRs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury. AU - Luyt, Karen AU - Slade, Timothy P AU - Dorward, Jienchi J AU - Durant, Claire F AU - Wu, Yue AU - Ryuichi Shigemoto AU - Mundell, Stuart J AU - Váradi, Anikó AU - Molnár, Elek ID - 2666 IS - 3 JF - Journal of Neurochemistry TI - Developing oligodendrocytes express functional GABAB receptors that stimulate cell proliferation and migration VL - 100 ER - TY - JOUR AB - Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are expressed postsynaptically in the rodent globus pallidus (GP), where they play several important roles in controlling GP neuronal activity. To further elucidate the role of HCN channels in the GP, immunocytochemical and electrophysiological approaches were used to test the hypothesis that HCN channels are also expressed presynaptically on the local axon collaterals of GP neurons. At the electron microscopic level, immunoperoxidase labelling for HCN1 and HCN2 was localized in GP somata and dendritic processes, myelinated and unmyelinated axons, and axon terminals. One population of labelled terminals formed symmetric synapses with somata and proximal dendrites and were immunoreactive for parvalbumin, consistent with the axon collaterals of GABAergic GP projection neurons. In addition, labelling for HCN2 and, to a lesser degree, HCN1 was observed in axon terminals that formed asymmetric synapses and were immunoreactive for the vesicular glutamate transporter 2. Immunogold labelling demonstrated that HCN1 and HCN2 were located predominantly at extrasynaptic sites along the plasma membrane of both types of terminal. To determine the function of presynaptic HCN channels in the GP, we performed whole-cell recordings from GP neurons in vitro. Bath application of the HCN channel blocker ZD7288 resulted in an increase in the frequency of mIPSCs but had no effect on their amplitude, implying that HCN channels tonically regulate the release of GABA. Their presence, and predicted role in modulating transmitter release, represents a hitherto unidentified mechanism whereby HCN channels influence the activity of GP neurons. AU - Boyes, Justin AU - Bolam, John P AU - Ryuichi Shigemoto AU - Stanford, Ian M ID - 2668 IS - 7 JF - European Journal of Neuroscience TI - Functional presynaptic HCN channels in the rat globus pallidus VL - 25 ER -