@article{12875, abstract = {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.}, author = {Cheung, Giselle T and Pauler, Florian and Koppensteiner, Peter and Krausgruber, Thomas and Streicher, Carmen and Schrammel, Martin and Özgen, Natalie Y and Ivec, Alexis and Bock, Christoph and Shigemoto, Ryuichi and Hippenmeyer, Simon}, issn = {0896-6273}, journal = {Neuron}, number = {2}, pages = {230--246.e11}, publisher = {Elsevier}, title = {{Multipotent progenitors instruct ontogeny of the superior colliculus}}, doi = {10.1016/j.neuron.2023.11.009}, volume = {112}, year = {2024}, } @article{15084, abstract = {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.}, author = {Koppensteiner, Peter and Bhandari, Pradeep and Önal, Hüseyin C and Borges Merjane, Carolina and Le Monnier, Elodie and Roy, Utsa and Nakamura, Yukihiro and Sadakata, Tetsushi and Sanbo, Makoto and Hirabayashi, Masumi and Rhee, JeongSeop and Brose, Nils and Jonas, Peter M and Shigemoto, Ryuichi}, issn = {1091-6490}, journal = {Proceedings of the National Academy of Sciences}, number = {8}, publisher = {Proceedings of the National Academy of Sciences}, title = {{GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles}}, doi = {10.1073/pnas.2301449121}, volume = {121}, year = {2024}, } @article{14843, abstract = {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.}, author = {Chen, JingJing and Kaufmann, Walter and Chen, Chong and Arai, Itaru and Kim, Olena and Shigemoto, Ryuichi and Jonas, Peter M}, issn = {1097-4199}, journal = {Neuron}, publisher = {Elsevier}, title = {{Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse}}, doi = {10.1016/j.neuron.2023.12.002}, year = {2024}, } @article{14253, abstract = {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.}, author = {Vierra, Nicholas C. and Ribeiro-Silva, Luisa and Kirmiz, Michael and Van Der List, Deborah and Bhandari, Pradeep and Mack, Olivia A. and Carroll, James and Le Monnier, Elodie and Aicher, Sue A. and Shigemoto, Ryuichi and Trimmer, James S.}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling}}, doi = {10.1038/s41467-023-40930-6}, volume = {14}, year = {2023}, } @article{13202, abstract = {Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons.}, author = {Eguchi, Kohgaku and Le Monnier, Elodie and Shigemoto, Ryuichi}, issn = {1529-2401}, journal = {The Journal of Neuroscience}, number = {23}, pages = {4197--4216}, publisher = {Society for Neuroscience}, title = {{Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons}}, doi = {10.1523/JNEUROSCI.1514-22.2023}, volume = {43}, year = {2023}, } @misc{13173, abstract = {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.}, author = {Shigemoto, Ryuichi}, keywords = {medial habenula, GABAB receptor, vesicle release, Flash and Freeze, Flash and Freeze-fracture}, publisher = {Institute of Science and Technology Austria}, title = {{Transition from tonic to phasic neurotransmitter release by presynaptic GABAB receptor activation in medial habenula terminals}}, doi = {10.15479/AT:ISTA:13173}, year = {2023}, } @article{10890, abstract = {Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission.}, author = {Eguchi, Kohgaku and Montanaro-Punzengruber, Jacqueline-Claire and Le Monnier, Elodie and Shigemoto, Ryuichi}, issn = {16625129}, journal = {Frontiers in Neuroanatomy}, publisher = {Frontiers}, title = {{The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals}}, doi = {10.3389/fnana.2022.846615}, volume = {16}, year = {2022}, } @article{10889, abstract = {Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.}, author = {Shigemoto, Ryuichi}, issn = {2050-5701}, journal = {Microscopy}, number = {Supplement_1}, pages = {i72--i80}, publisher = {Oxford Academic}, title = {{Electron microscopic visualization of single molecules by tag-mediated metal particle labeling}}, doi = {10.1093/jmicro/dfab048}, volume = {71}, year = {2022}, } @article{12212, abstract = {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.}, author = {Martín-Belmonte, Alejandro and Aguado, Carolina and Alfaro-Ruiz, Rocío and Moreno-Martínez, Ana Esther and de la Ossa, Luis and Aso, Ester and Gómez-Acero, Laura and Shigemoto, Ryuichi and Fukazawa, Yugo and Ciruela, Francisco and Luján, Rafael}, issn = {1758-9193}, journal = {Alzheimer's Research & Therapy}, keywords = {Cognitive Neuroscience, Neurology (clinical), Neurology}, publisher = {Springer Nature}, title = {{Nanoscale alterations in GABAB receptors and GIRK channel organization on the hippocampus of APP/PS1 mice}}, doi = {10.1186/s13195-022-01078-5}, volume = {14}, year = {2022}, } @article{7551, abstract = {Novelty facilitates formation of memories. The detection of novelty and storage of contextual memories are both mediated by the hippocampus, yet the mechanisms that link these two functions remain to be defined. Dentate granule cells (GCs) of the dorsal hippocampus fire upon novelty exposure forming engrams of contextual memory. However, their key excitatory inputs from the entorhinal cortex are not responsive to novelty and are insufficient to make dorsal GCs fire reliably. Here we uncover a powerful glutamatergic pathway to dorsal GCs from ventral hippocampal mossy cells (MCs) that relays novelty, and is necessary and sufficient for driving dorsal GCs activation. Furthermore, manipulation of ventral MCs activity bidirectionally regulates novelty-induced contextual memory acquisition. Our results show that ventral MCs activity controls memory formation through an intra-hippocampal interaction mechanism gated by novelty.}, author = {Fredes Tolorza, Felipe A and Silva Sifuentes, Maria A and Koppensteiner, Peter and Kobayashi, Kenta and Jösch, Maximilian A and Shigemoto, Ryuichi}, journal = {Current Biology}, number = {1}, pages = {P25--38.E5}, publisher = {Elsevier}, title = {{Ventro-dorsal hippocampal pathway gates novelty-induced contextual memory formation}}, doi = {10.1016/j.cub.2020.09.074}, volume = {31}, year = {2021}, } @article{9330, abstract = {In nerve cells the genes encoding for α2δ subunits of voltage-gated calcium channels have been linked to synaptic functions and neurological disease. Here we show that α2δ subunits are essential for the formation and organization of glutamatergic synapses. Using a cellular α2δ subunit triple-knockout/knockdown model, we demonstrate a failure in presynaptic differentiation evidenced by defective presynaptic calcium channel clustering and calcium influx, smaller presynaptic active zones, and a strongly reduced accumulation of presynaptic vesicle-associated proteins (synapsin and vGLUT). The presynaptic defect is associated with the downscaling of postsynaptic AMPA receptors and the postsynaptic density. The role of α2δ isoforms as synaptic organizers is highly redundant, as each individual α2δ isoform can rescue presynaptic calcium channel trafficking and expression of synaptic proteins. Moreover, α2δ-2 and α2δ-3 with mutated metal ion-dependent adhesion sites can fully rescue presynaptic synapsin expression but only partially calcium channel trafficking, suggesting that the regulatory role of α2δ subunits is independent from its role as a calcium channel subunit. Our findings influence the current view on excitatory synapse formation. First, our study suggests that postsynaptic differentiation is secondary to presynaptic differentiation. Second, the dependence of presynaptic differentiation on α2δ implicates α2δ subunits as potential nucleation points for the organization of synapses. Finally, our results suggest that α2δ subunits act as transsynaptic organizers of glutamatergic synapses, thereby aligning the synaptic active zone with the postsynaptic density.}, author = {Schöpf, Clemens L. and Ablinger, Cornelia and Geisler, Stefanie M. and Stanika, Ruslan I. and Campiglio, Marta and Kaufmann, Walter and Nimmervoll, Benedikt and Schlick, Bettina and Brockhaus, Johannes and Missler, Markus and Shigemoto, Ryuichi and Obermair, Gerald J.}, issn = {1091-6490}, journal = {PNAS}, number = {14}, publisher = {National Academy of Sciences}, title = {{Presynaptic α2δ subunits are key organizers of glutamatergic synapses}}, doi = {10.1073/pnas.1920827118}, volume = {118}, year = {2021}, } @article{9641, abstract = {At the encounter with a novel environment, contextual memory formation is greatly enhanced, accompanied with increased arousal and active exploration. Although this phenomenon has been widely observed in animal and human daily life, how the novelty in the environment is detected and contributes to contextual memory formation has lately started to be unveiled. The hippocampus has been studied for many decades for its largely known roles in encoding spatial memory, and a growing body of evidence indicates a differential involvement of dorsal and ventral hippocampal divisions in novelty detection. In this brief review article, we discuss the recent findings of the role of mossy cells in the ventral hippocampal moiety in novelty detection and put them in perspective with other novelty-related pathways in the hippocampus. We propose a mechanism for novelty-driven memory acquisition in the dentate gyrus by the direct projection of ventral mossy cells to dorsal dentate granule cells. By this projection, the ventral hippocampus sends novelty signals to the dorsal hippocampus, opening a gate for memory encoding in dentate granule cells based on information coming from the entorhinal cortex. We conclude that, contrary to the presently accepted functional independence, the dorsal and ventral hippocampi cooperate to link the novelty and contextual information, and this dorso-ventral interaction is crucial for the novelty-dependent memory formation.}, author = {Fredes, Felipe and Shigemoto, Ryuichi}, issn = {10959564}, journal = {Neurobiology of Learning and Memory}, publisher = {Elsevier}, title = {{The role of hippocampal mossy cells in novelty detection}}, doi = {10.1016/j.nlm.2021.107486}, volume = {183}, year = {2021}, } @article{10051, abstract = {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.}, author = {Butola, Tanvi and Alvanos, Theocharis and Hintze, Anika and Koppensteiner, Peter and Kleindienst, David and Shigemoto, Ryuichi and Wichmann, Carolin and Moser, Tobias}, issn = {1529-2401}, journal = {Journal of Neuroscience}, number = {37}, pages = {7742--7767}, publisher = {Society for Neuroscience}, title = {{RIM-binding protein 2 organizes Ca21 channel topography and regulates release probability and vesicle replenishment at a fast central synapse}}, doi = {10.1523/JNEUROSCI.0586-21.2021}, volume = {41}, year = {2021}, } @article{10403, abstract = {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.}, author = {Biane, Celia and Rückerl, Florian and Abrahamsson, Therese and Saint-Cloment, Cécile and Mariani, Jean and Shigemoto, Ryuichi and Digregorio, David A. and Sherrard, Rachel M. and Cathala, Laurence}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Developmental emergence of two-stage nonlinear synaptic integration in cerebellar interneurons}}, doi = {10.7554/eLife.65954}, volume = {10}, year = {2021}, } @article{9437, abstract = {The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.}, author = {Bhandari, Pradeep and Vandael, David H and Fernández-Fernández, Diego and Fritzius, Thorsten and Kleindienst, David and Önal, Hüseyin C and Montanaro-Punzengruber, Jacqueline-Claire and Gassmann, Martin and Jonas, Peter M and Kulik, Akos and Bettler, Bernhard and Shigemoto, Ryuichi and Koppensteiner, Peter}, issn = {2050-084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals}}, doi = {10.7554/ELIFE.68274}, volume = {10}, year = {2021}, } @inbook{9756, abstract = {High-resolution visualization and quantification of membrane proteins contribute to the understanding of their functions and the roles they play in physiological and pathological conditions. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is a powerful electron microscopy method to study quantitatively the two-dimensional distribution of transmembrane proteins and their tightly associated proteins. During treatment with SDS, intracellular organelles and proteins not anchored to the replica are dissolved, whereas integral membrane proteins captured and stabilized by carbon/platinum deposition remain on the replica. Their intra- and extracellular domains become exposed on the surface of the replica, facilitating the accessibility of antibodies and, therefore, providing higher labeling efficiency than those obtained with other immunoelectron microscopy techniques. In this chapter, we describe the protocols of SDS-FRL adapted for mammalian brain samples, and optimization of the SDS treatment to increase the labeling efficiency for quantification of Cav2.1, the alpha subunit of P/Q-type voltage-dependent calcium channels utilizing deep learning algorithms.}, author = {Kaufmann, Walter and Kleindienst, David and Harada, Harumi and Shigemoto, Ryuichi}, booktitle = { Receptor and Ion Channel Detection in the Brain}, isbn = {9781071615218}, keywords = {Freeze-fracture replica: Deep learning, Immunogold labeling, Integral membrane protein, Electron microscopy}, pages = {267--283}, publisher = {Humana}, title = {{High-Resolution localization and quantitation of membrane proteins by SDS-digested freeze-fracture replica labeling (SDS-FRL)}}, doi = {10.1007/978-1-0716-1522-5_19}, volume = {169}, year = {2021}, } @article{7148, abstract = {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.}, author = {Nakamoto, Chihiro and Konno, Kohtarou and Miyazaki, Taisuke and Nakatsukasa, Ena and Natsume, Rie and Abe, Manabu and Kawamura, Meiko and Fukazawa, Yugo and Shigemoto, Ryuichi and Yamasaki, Miwako and Sakimura, Kenji and Watanabe, Masahiko}, issn = {1096-9861}, journal = {Journal of Comparative Neurology}, number = {6}, pages = {1003--1027}, publisher = {Wiley}, title = {{Expression mapping, quantification, and complex formation of GluD1 and GluD2 glutamate receptors in adult mouse brain}}, doi = {10.1002/cne.24792}, volume = {528}, year = {2020}, } @article{7664, abstract = {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}, author = {Martín-Belmonte, Alejandro and Aguado, Carolina and Alfaro-Ruíz, Rocío and Moreno-Martínez, Ana Esther and De La Ossa, Luis and Martínez-Hernández, José and Buisson, Alain and Shigemoto, Ryuichi and Fukazawa, Yugo and Luján, Rafael}, issn = {14220067}, journal = {International journal of molecular sciences}, number = {7}, publisher = {MDPI}, title = {{Density of GABAB receptors is reduced in granule cells of the hippocampus in a mouse model of Alzheimer's disease}}, doi = {10.3390/ijms21072459}, volume = {21}, year = {2020}, } @article{7665, abstract = {Acute brain slice preparation is a powerful experimental model for investigating the characteristics of synaptic function in the brain. Although brain tissue is usually cut at ice-cold temperature (CT) to facilitate slicing and avoid neuronal damage, exposure to CT causes molecular and architectural changes of synapses. To address these issues, we investigated ultrastructural and electrophysiological features of synapses in mouse acute cerebellar slices prepared at ice-cold and physiological temperature (PT). In the slices prepared at CT, we found significant spine loss and reconstruction, synaptic vesicle rearrangement and decrease in synaptic proteins, all of which were not detected in slices prepared at PT. Consistent with these structural findings, slices prepared at PT showed higher release probability. Furthermore, preparation at PT allows electrophysiological recording immediately after slicing resulting in higher detectability of long-term depression (LTD) after motor learning compared with that at CT. These results indicate substantial advantages of the slice preparation at PT for investigating synaptic functions in different physiological conditions.}, author = {Eguchi, Kohgaku and Velicky, Philipp and Hollergschwandtner, Elena and Itakura, Makoto and Fukazawa, Yugo and Danzl, Johann G and Shigemoto, Ryuichi}, issn = {16625102}, journal = {Frontiers in Cellular Neuroscience}, publisher = {Frontiers Media}, title = {{Advantages of acute brain slices prepared at physiological temperature in the characterization of synaptic functions}}, doi = {10.3389/fncel.2020.00063}, volume = {14}, year = {2020}, } @article{7878, abstract = {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.}, author = {Bao, Jin and Graupner, Michael and Astorga, Guadalupe and Collin, Thibault and Jalil, Abdelali and Indriati, Dwi Wahyu and Bradley, Jonathan and Shigemoto, Ryuichi and Llano, Isabel}, issn = {2050084X}, journal = {eLife}, publisher = {eLife Sciences Publications}, title = {{Synergism of type 1 metabotropic and ionotropic glutamate receptors in cerebellar molecular layer interneurons in vivo}}, doi = {10.7554/eLife.56839}, volume = {9}, year = {2020}, }