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 - JOUR AB - Mapping the complex and dense arrangement of cells and their connectivity in brain tissue demands nanoscale spatial resolution imaging. Super-resolution optical microscopy excels at visualizing specific molecules and individual cells but fails to provide tissue context. Here we developed Comprehensive Analysis of Tissues across Scales (CATS), a technology to densely map brain tissue architecture from millimeter regional to nanometer synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS uses fixation-compatible extracellular labeling and optical imaging, including stimulated emission depletion or expansion microscopy, to comprehensively delineate cellular structures. It enables three-dimensional reconstruction of single synapses and mapping of synaptic connectivity by identification and analysis of putative synaptic cleft regions. Applying CATS to the mouse hippocampal mossy fiber circuitry, we reconstructed and quantified the synaptic input and output structure of identified neurons. We furthermore demonstrate applicability to clinically derived human tissue samples, including formalin-fixed paraffin-embedded routine diagnostic specimens, for visualizing the cellular architecture of brain tissue in health and disease. AU - Michalska, Julia M AU - Lyudchik, Julia AU - Velicky, Philipp AU - Korinkova, Hana AU - Watson, Jake AU - Cenameri, Alban AU - Sommer, Christoph M AU - Amberg, Nicole AU - Venturino, Alessandro AU - Roessler, Karl AU - Czech, Thomas AU - Höftberger, Romana AU - Siegert, Sandra AU - Novarino, Gaia AU - Jonas, Peter M AU - Danzl, Johann G ID - 14257 JF - Nature Biotechnology SN - 1087-0156 TI - Imaging brain tissue architecture across millimeter to nanometer scales 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 - Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer’s disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10–20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission. AU - Hori, Tetsuya AU - Eguchi, Kohgaku AU - Wang, Han Ying AU - Miyasaka, Tomohiro AU - Guillaud, Laurent AU - Taoufiq, Zacharie AU - Mahapatra, Satyajit AU - Yamada, Hiroshi AU - Takei, Kohji AU - Takahashi, Tomoyuki ID - 11419 JF - eLife TI - Microtubule assembly by tau impairs endocytosis and neurotransmission via dynamin sequestration in Alzheimer's disease synapse model VL - 11 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 - Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site. AU - Thayyil, Sampreeth AU - Nishigami, Yukinori AU - Islam, Muhammad J AU - Hashim, P. K. AU - Furuta, Ken'Ya AU - Oiwa, Kazuhiro AU - Yu, Jian AU - Yao, Min AU - Nakagaki, Toshiyuki AU - Tamaoki, Nobuyuki ID - 11333 IS - 30 JF - Chemistry - A European Journal SN - 09476539 TI - Dynamic control of microbial movement by photoswitchable ATP antagonists VL - 28 ER - TY - THES AB - AMPA receptors (AMPARs) mediate fast excitatory neurotransmission and their role is implicated in complex processes such as learning and memory and various neurological diseases. These receptors are composed of different subunits and the subunit composition can affect channel properties, receptor trafficking and interaction with other associated proteins. Using the high sensitivity SDS-digested freeze-fracture replica labeling (SDS-FRL) for electron microscopy I investigated the number, density, and localization of AMPAR subunits, GluA1, GluA2, GluA3, and GluA1-3 (panAMPA) in pyramidal cells in the CA1 area of mouse hippocampus. I have found that the immunogold labeling for all of these subunits in the postsynaptic sites was highest in stratum radiatum and lowest in stratum lacunosummoleculare. The labeling density for the all subunits in the extrasynaptic sites showed a gradual increase from the pyramidal cell soma towards the distal part of stratum radiatum. The densities of extrasynaptic GluA1, GluA2 and panAMPA labeling reached 10-15% of synaptic densities, while the ratio of extrasynaptic labeling for GluA3 was significantly lower compared than those for other subunits. The labeling patterns for GluA1, GluA2 and GluA1-3 are similar and their densities were higher in the periphery than center of synapses. In contrast, the GluA3- containing receptors were more centrally localized compared to the GluA1- and GluA2- containing receptors. The hippocampus plays a central role in learning and memory. Contextual learning has been shown to require the delivery of AMPA receptors to CA1 synapses in the dorsal hippocampus. However, proximodistal heterogeneity of this plasticity and particular contribution of different AMPA receptor subunits are not fully understood. By combining inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, with SDS-FRL, I have revealed an increase in synaptic density specific to GluA1-containing AMPA receptors in the CA1 area. The intrasynaptic distribution of GluA1 also changed from the periphery to center-preferred pattern. Furthermore, this synaptic plasticity was evident selectively in stratum radiatum but not stratum oriens, and in the CA1 subregion proximal but not distal to CA2. These findings further contribute to our understanding of how specific hippocampal subregions and AMPA receptor subunits are involved in physiological learning. Although the immunolabeling results above shed light on subunit-specific plasticity in AMPAR distribution, no tools to visualize and study the subunit composition at the single channel level in situ have been available. Electron microscopy with conventional immunogold labeling approaches has limitations in the single channel analysis because of the large size of antibodies and steric hindrance hampering multiple subunit labeling of single channels. I managed to develop a new chemical labeling system using a short peptide tag and small synthetic probes, which form specific covalent bond with a cysteine residue in the tag fused to proteins of interest (reactive tag system). I additionally made substantial progress into adapting this system for AMPA receptor subunits. AU - Jevtic, Marijo ID - 11393 SN - 2663-337X TI - Contextual fear learning induced changes in AMPA receptor subtypes along the proximodistal axis in dorsal hippocampus 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 - THES AB - Left-right asymmetries can be considered a fundamental organizational principle of the vertebrate central nervous system. The hippocampal CA3-CA1 pyramidal cell synaptic connection shows an input-side dependent asymmetry where the hemispheric location of the presynaptic CA3 neuron determines the synaptic properties. Left-input synapses terminating on apical dendrites in stratum radiatum have a higher density of NMDA receptor subunit GluN2B, a lower density of AMPA receptor subunit GluA1 and smaller areas with less often perforated PSDs. On the other hand, left-input synapses terminating on basal dendrites in stratum oriens have lower GluN2B densities than right-input ones. Apical and basal synapses further employ different signaling pathways involved in LTP. SDS-digested freeze-fracture replica labeling can visualize synaptic membrane proteins with high sensitivity and resolution, and has been used to reveal the asymmetry at the electron microscopic level. However, it requires time-consuming manual demarcation of the synaptic surface for quantitative measurements. To facilitate the analysis of replica labeling, I first developed a software named Darea, which utilizes deep-learning to automatize this demarcation. With Darea I characterized the synaptic distribution of NMDA and AMPA receptors as well as the voltage-gated Ca2+ channels in CA1 stratum radiatum and oriens. Second, I explored the role of GluN2B and its carboxy-terminus in the establishment of input-side dependent hippocampal asymmetry. In conditional knock-out mice lacking GluN2B expression in CA1 and GluN2B-2A swap mice, where GluN2B carboxy-terminus was exchanged to that of GluN2A, no significant asymmetries of GluN2B, GluA1 and PSD area were detected. We further discovered a previously unknown functional asymmetry of GluN2A, which was also lost in the swap mouse. These results demonstrate that GluN2B carboxy-terminus plays a critical role in normal formation of input-side dependent asymmetry. AU - Kleindienst, David ID - 9562 SN - 2663-337X TI - 2B or not 2B: Hippocampal asymmetries mediated by NMDA receptor subunit GluN2B C-terminus and high-throughput image analysis by Deep-Learning 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 - Cytoskeletal filaments such as microtubules (MTs) and filamentous actin (F-actin) dynamically support cell structure and functions. In central presynaptic terminals, F-actin is expressed along the release edge and reportedly plays diverse functional roles, but whether axonal MTs extend deep into terminals and play any physiological role remains controversial. At the calyx of Held in rats of either sex, confocal and high-resolution microscopy revealed that MTs enter deep into presynaptic terminal swellings and partially colocalize with a subset of synaptic vesicles (SVs). Electrophysiological analysis demonstrated that depolymerization of MTs specifically prolonged the slow-recovery time component of EPSCs from short-term depression induced by a train of high-frequency stimulation, whereas depolymerization of F-actin specifically prolonged the fast-recovery component. In simultaneous presynaptic and postsynaptic action potential recordings, depolymerization of MTs or F-actin significantly impaired the fidelity of high-frequency neurotransmission. We conclude that MTs and F-actin differentially contribute to slow and fast SV replenishment, thereby maintaining high-frequency neurotransmission. AU - Piriya Ananda Babu, Lashmi AU - Wang, Han Ying AU - Eguchi, Kohgaku AU - Guillaud, Laurent AU - Takahashi, Tomoyuki ID - 7339 IS - 1 JF - Journal of neuroscience TI - Microtubule and actin differentially regulate synaptic vesicle cycling to maintain high-frequency neurotransmission VL - 40 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 -