@article{21502, abstract = {The mammalian brain stores glucose, the main circulating energy substrate, as glycogen. In rodents, the cerebellum contains relatively high glycogen levels, yet its cellular and subcellular distribution remains poorly defined. Using monoclonal antibodies against glycogen, we examined its distribution in the mouse cerebellar cortex. Glycogen was predominantly localized to Bergmann glia (BG) processes in the molecular layer and was also detected in Purkinje cells (PCs), the principal cerebellar neurons. To assess the functional significance of cerebellar glycogen, we analyzed behavior in mice lacking glycogen synthase 1 (Gys1) in BG or PCs using a floxed Gys1 line. Gys1 deficiency in either PCs or GFAP-positive cells reduced anxiety-like behavior, whereas combined deletion caused PC degeneration and ataxia. These findings reveal a critical role for glycogen metabolism in both astrocytes and neurons in cerebellar function.}, author = {Akther, Sonam and Lee, Ashley Bomin and Konno, Ayumu and Asiminas, Antonis and Vittani, Marta and Mishima, Tsuneko and Hirai, Hirokazu and Meehan, Claire Francesca and Duran, Jordi and Guinovart, Joan and Ashida, Hitoshi and Morita, Tsuyoshi and Baba, Otto and Shigemoto, Ryuichi and Nedergaard, Maiken and Hirase, Hajime}, issn = {2589-0042}, journal = {iScience}, number = {4}, publisher = {Elsevier}, title = {{Distribution and functional significance of rodent cerebellar glycogen}}, doi = {10.1016/j.isci.2026.115192}, volume = {29}, year = {2026}, } @article{20659, abstract = {Metabotropic GABA (GABAB) receptors have modulatory functions on neuronal excitability and neurotransmitter release. To fulfil these functions, GABAB receptors form macromolecular signaling complexes with G proteins, effectors, and other associated proteins. Here we investigated the postnatal development of GABAB receptors (GABAB1 and GABAB2 subunits) in mouse brain, focusing on potential similarities in the spatial and temporal expression pattern of their associated proteins CaV2.1, Gαo, Gβ5, and RGS7, using histoblots, immunofluorescence, and immunoelectron microscopic techniques. At all ages analyzed, histoblot showed that the six proteins were widely expressed in the brain, with mostly an overlapping pattern throughout postnatal development. In the hippocampus, immunoelectron microscopy and quantitative analysis of immunoparticles for GABAB1, GABAB2, Gαo, Gβ5, and RGS7 revealed their progressive enrichment around excitatory synapses on dendritic spines of CA1 pyramidal cells toward P15. At presynaptic sites, GABAB receptors colocalize with CaV2.1, Gαo, Gβ5, and RGS7 in the active zone and extrasynaptic membranes of axon terminals, establishing synapses on dendritic spines of CA1 pyramidal cells. In the cerebellum, double immunofluorescence at P7 and P10 revealed the colocalization of GABAB1 and CaV2.1 in the whole dendritic tree of developing Purkinje cells. Immunoelectron microscopy at P15 showed that GABAB1, GABAB2, CaV2.1, Gαo, Gβ5, and RGS7 are distributed along the dendritic surface of Purkinje cells, enriched close to excitatory synapses in spines. Altogether, these data suggest that macromolecular complexes composed of GABAB1 /GABAB2/CaV2.1/ Gαo/Gβ5/RGS7 are pre-assembled during key stages of postnatal development in hippocampal and cerebellar neurons.}, author = {Aguado, Carolina and Alfaro-Ruiz, Rocío and Martínez-Poyato, María Llanos and Moreno-Martínez, Ana Esther and García-Madrona, Sebastián and Roldán-Sastre, Alberto and Alonso-Gómez, Pablo and Fernández, Miriam and Puertas-Avendaño, Ricardo and Shigemoto, Ryuichi and Martemyanov, Kirill A. and Luján, Rafael}, issn = {1699-5848}, journal = {Histology and Histopathology}, number = {12}, pages = {1967--1984}, publisher = {Sercrisma International}, title = {{Developmental regulation of GABAB receptors and downstream molecules in the mouse brain}}, doi = {10.14670/HH-18-970}, volume = {40}, year = {2025}, } @article{17293, abstract = {Voltage-gated CaV2.1 (P/Q-type) Ca2+ channels play a crucial role in regulating neurotransmitter release, thus contributing to synaptic plasticity and to processes such as learning and memory. Despite their recognized importance in neural function, there is limited information on their potential involvement in neurodegenerative conditions such as Alzheimer's disease (AD). Here, we aimed to explore the impact of AD pathology on the density and nanoscale compartmentalization of CaV2.1 channels in the hippocampus in association with GABAB receptors. Histoblotting experiments showed that the density of CaV2.1 channel was significantly reduced in the hippocampus of APP/PS1 mice in a laminar-dependent manner. CaV2.1 channel was enriched in the active zone of the axon terminals and was present at a very low density over the surface of dendritic tree of the CA1 pyramidal cells, as shown by quantitative SDS-digested freeze-fracture replica labelling (SDS-FRL). In APP/PS1 mice, the density of CaV2.1 channel in the active zone was significantly reduced in the strata radiatum and lacunosum-moleculare, while it remained unaltered in the stratum oriens. The decline in Cav2.1 channel density was found to be associated with a corresponding impairment in the GABAergic synaptic function, as evidenced by electrophysiological experiments carried out in the hippocampus of APP/PS1 mice. Remarkably, double SDS-FRL showed a co-clustering of CaV2.1 channel and GABAB1 receptor in nanodomains (~40–50 nm) in wild type mice, while in APP/PS1 mice this nanoarchitecture was absent. Together, these findings suggest that the AD pathology-induced reduction in CaV2.1 channel density and CaV2.1-GABAB1 de-clustering may play a role in the synaptic transmission alterations shown in the AD hippocampus. Therefore, uncovering these layer-dependent changes in P/Q calcium currents associated with AD pathology can benefit the development of future strategies for AD management.}, author = {Martín‐Belmonte, Alejandro and Aguado, Carolina and Alfaro‐Ruiz, Rocío and Kulik, Akos and de la Ossa, Luis and Moreno‐Martínez, Ana Esther and Alberquilla, Samuel and García‐Carracedo, Lucía and Fernández, Miriam and Fajardo‐Serrano, Ana and Aso, Ester and Shigemoto, Ryuichi and Martín, Eduardo D. and Fukazawa, Yugo and Ciruela, Francisco and Luján, Rafael}, issn = {1750-3639}, journal = {Brain Pathology}, number = {2}, publisher = {Wiley}, title = {{Nanoarchitecture of CaV>2.1 channels and GABAB receptors in the mouse hippocampus: Impact of APP/PS1 pathology}}, doi = {10.1111/bpa.13279}, volume = {35}, year = {2025}, } @phdthesis{19271, abstract = {The medial habenula (MHb) is implicated in regulating emotional responses to aversive events. Studies in zebrafish have identified a remarkable morphological left-right asymmetry in the dorsal habenula (zebrafish equivalent of mammalian MHb)-to-interpeduncular nucleus (IPN) pathway and its left-side specific role in modulating fear responses. However, there is little evidence for structural or functional lateralization in the mammalian MHb-IPN pathway. Here, I investigated the synaptic properties of the left and right MHb afferents to the IPN in mice and addressed whether these synaptic connections selectively influence the expression of conditioned fear in mice. My findings reveal that each individual IPN neuron receives inputs from both left and right MHb. Electrophysiological recordings from the same postsynaptic IPN neurons demonstrate that the left MHb-originating synapses exhibit lower release probability and higher 𝛾-aminobutyric acid type B receptor (GABABR)-mediated potentiation compared to the right MHb-originating synapses. Interestingly, chemogenetic inhibition of cholinergic neurons in the left but not the right MHb significantly attenuated cue-dependent fear recall. Furthermore, conditional deletion of GABABR in the left MHb interfered with the recall of cued fear memory, whereas that in the right MHb neurons spared fear memory expression. Collectively, I demonstrate a functional asymmetry of the MHb in mice, revealing a predominant role for GABABR-mediated signaling in the left MHb-IPN pathway in the modulation of fear memories. These findings suggest that lateralized pathways could represent a fundamental principle in the neural regulation of emotion across species.}, author = {Önal, Hüseyin C}, issn = {2663-337X}, publisher = {Institute of Science and Technology Austria}, title = {{Asymmetrical modulation of fear expression via GABAB receptors in the mouse medial habenula}}, doi = {10.15479/AT-ISTA-19271}, year = {2025}, } @inbook{18052, abstract = {Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) is an electron microscope (EM) sample preparation technique which allows for high-resolution visualization of membrane proteins with high sensitivity. However, image acquisition of specific replica profiles such as synapses in a large field of EM view needs a valid experience and a long time for manual searching. Here, we describe how to utilize deep learning for automatizing image acquisition of specific profiles of interest in replica samples. This protocol facilitates the labor-intensive collection of EM images, in particular for rare profiles. We provide instructions for using SerialEM image acquisition software in conjunction with object detection by our newly developed deep learning software DarEM, to automatically acquire tilt series of all synapses in a selected region. We then show how to perform a mostly automated analysis of gold particle labeling in the acquired images by utilizing Darea software.}, author = {Kleindienst, David and Costanzo, Tommaso and Shigemoto, Ryuichi}, booktitle = {New Aspects in Analyzing the Synaptic Organization of the Brain}, editor = {Lübke, Joachim H.R. and Rollenhagen, Astrid}, isbn = {9781071640180}, issn = {1940-6045}, pages = {123--137}, publisher = {Springer Nature}, title = {{Automated Imaging and Analysis of Synapses in Freeze-Fracture Replica Samples with Deep Learning}}, doi = {10.1007/978-1-0716-4019-7_8}, year = {2024}, } @article{18445, abstract = {Acquisition of specialized cellular features is controlled by the ordered expression of transcription factors (TFs) along differentiation trajectories. Here, we find a member of the Onecut TF family, ONECUT3, expressed in postmitotic neurons that leave their Ascl1+/Onecut1/2+ proliferative domain in the vertebrate hypothalamus to instruct neuronal differentiation. We combined single-cell RNA-seq and gain-of-function experiments for gene network reconstruction to show that ONECUT3 affects the polarization and morphogenesis of both hypothalamic GABA-derived dopamine and thyrotropin-releasing hormone (TRH)+ glutamate neurons through neuron navigator-2 (NAV2). In vivo, siRNA-mediated knockdown of ONECUT3 in neonatal mice reduced NAV2 mRNA, as well as neurite complexity in Onecut3-containing neurons, while genetic deletion of Onecut3/ceh-48 in C. elegans impaired neurocircuit wiring, and sensory discrimination-based behaviors. Thus, ONECUT3, conserved across neuronal subtypes and many species, underpins the polarization and morphological plasticity of phenotypically distinct neurons that descend from a common pool of Ascl1+ progenitors in the hypothalamus.}, author = {Zupančič, Maja and Keimpema, Erik and Tretiakov, Evgenii O. and Eder, Stephanie J. and Lev, Itamar and Englmaier, Lukas and Bhandari, Pradeep and Fietz, Simone A. and Härtig, Wolfgang and Renaux, Estelle and Villunger, Andreas and Hökfelt, Tomas and Zimmer, Manuel and Clotman, Frédéric and Harkany, Tibor}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Concerted transcriptional regulation of the morphogenesis of hypothalamic neurons by ONECUT3}}, doi = {10.1038/s41467-024-52762-z}, volume = {15}, year = {2024}, } @misc{15385, abstract = {Relevant information about the data can be found in the 'Readme_Data.txt' file. A previous version of the publication can be found on BioRxiv: https://www.biorxiv.org/content/10.1101/2022.10.11.511691v4 and published in Plos Biology (2024)}, author = {Burnett, Laura and Koppensteiner, Peter and Symonova, Olga and Masson, Tomas and Vega Zuniga, Tomas A and Contreras, Ximena and Rülicke, Thomas and Shigemoto, Ryuichi and Novarino, Gaia and Jösch, Maximilian A}, keywords = {ASD, periaqueductal gray, perception, behavior, potassium channels}, publisher = {Institute of Science and Technology Austria}, title = {{Shared behavioural impairments in visual perception and place avoidance across different autism models are driven by periaqueductal grey hypoexcitability in Setd5 haploinsufficient mice}}, doi = {10.15479/AT:ISTA:15385}, year = {2024}, } @article{17142, abstract = {Despite the diverse genetic origins of autism spectrum disorders (ASDs), affected individuals share strikingly similar and correlated behavioural traits that include perceptual and sensory processing challenges. Notably, the severity of these sensory symptoms is often predictive of the expression of other autistic traits. However, the origin of these perceptual deficits remains largely elusive. Here, we show a recurrent impairment in visual threat perception that is similarly impaired in 3 independent mouse models of ASD with different molecular aetiologies. Interestingly, this deficit is associated with reduced avoidance of threatening environments—a nonperceptual trait. Focusing on a common cause of ASDs, the Setd5 gene mutation, we define the molecular mechanism. We show that the perceptual impairment is caused by a potassium channel (Kv1)-mediated hypoexcitability in a subcortical node essential for the initiation of escape responses, the dorsal periaqueductal grey (dPAG). Targeted pharmacological Kv1 blockade rescued both perceptual and place avoidance deficits, causally linking seemingly unrelated trait deficits to the dPAG. Furthermore, we show that different molecular mechanisms converge on similar behavioural phenotypes by demonstrating that the autism models Cul3 and Ptchd1, despite having similar behavioural phenotypes, differ in their functional and molecular alteration. Our findings reveal a link between rapid perception controlled by subcortical pathways and appropriate learned interactions with the environment and define a nondevelopmental source of such deficits in ASD.}, author = {Burnett, Laura and Koppensteiner, Peter and Symonova, Olga and Masson, Tomas and Vega Zuniga, Tomas A and Contreras, Ximena and Rülicke, Thomas and Shigemoto, Ryuichi and Novarino, Gaia and Jösch, Maximilian A}, issn = {1545-7885}, journal = {PLoS Biology}, publisher = {Public Library of Science}, title = {{Shared behavioural impairments in visual perception and place avoidance across different autism models are driven by periaqueductal grey hypoexcitability in Setd5 haploinsufficient mice}}, doi = {10.1371/journal.pbio.3002668}, volume = {22}, year = {2024}, } @article{17280, abstract = {Adherens junction–associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1−/− and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.}, author = {Früh, Simon and Boudkkazi, Sami and Koppensteiner, Peter and Sereikaite, Vita and Chen, Li Yuan and Fernandez-Fernandez, Diego and Rem, Pascal D. and Ulrich, Daniel and Schwenk, Jochen and Chen, Ziyang and Monnier, Elodie Le and Fritzius, Thorsten and Innocenti, Sabrina M. and Besseyrias, Valérie and Trovò, Luca and Stawarski, Michal and Argilli, Emanuela and Sherr, Elliott H. and Van Bon, Bregje and Kamsteeg, Erik Jan and Iascone, Maria and Pilotta, Alba and Cutrì, Maria R. and Azamian, Mahshid S. and Hernández-García, Andrés and Lalani, Seema R. and Rosenfeld, Jill A. and Zhao, Xiaonan and Vogel, Tiphanie P. and Ona, Herda and Scott, Daryl A. and Scheiffele, Peter and Strømgaard, Kristian and Tafti, Mehdi and Gassmann, Martin and Fakler, Bernd and Shigemoto, Ryuichi and Bettler, Bernhard}, issn = {2375-2548}, journal = {Science Advances}, number = {28}, publisher = {American Association for the Advancement of Science}, title = {{Monoallelic de novo AJAP1 loss-of- function variants disrupt trans-synaptic control of neurotransmitter release}}, doi = {10.1126/sciadv.adk5462}, volume = {10}, year = {2024}, } @article{17457, abstract = {Autoantibodies against the protein leucine-rich glioma inactivated 1 (LGI1) cause the most common subtype of autoimmune encephalitis with predominant involvement of the limbic system, associated with seizures and memory deficits. LGI1 and its receptor ADAM22 are part of a transsynaptic protein complex that includes several proteins involved in presynaptic neurotransmitter release and postsynaptic glutamate sensing. Autoantibodies against LGI1 increase excitatory synaptic strength, but studies that genetically disrupt the LGI1-ADAM22 complex report a reduction in postsynaptic glutamate receptor-mediated responses. Thus, the mechanisms underlying the increased synaptic strength induced by LGI1 autoantibodies remain elusive, and the contributions of presynaptic molecules to the LGI1-transsynaptic complex remain unclear. We therefore investigated the presynaptic mechanisms that mediate autoantibody-induced synaptic strengthening.}, author = {Ritzau-Jost, Andreas and Gsell, Felix and Sell, Josefine and Sachs, Stefan and Montanaro-Punzengruber, Jacqueline-Claire and Kirmann, Toni and Maaß, Sebastian and Irani, Sarosh R. and Werner, Christian and Geis, Christian and Sauer, Markus and Shigemoto, Ryuichi and Hallermann, Stefan}, issn = {2332-7812}, journal = {Neurology, Neuroimmunology and Neuroinflammation}, number = {5}, pages = {e200284}, publisher = {Wolters Kluwer}, title = {{LGI1 autoantibodies enhance synaptic transmission by presynaptic Kv1 loss and increased action potential broadening}}, doi = {10.1212/NXI.0000000000200284}, volume = {11}, year = {2024}, } @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{14257, abstract = {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.}, author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Amberg, Nicole and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Höftberger, Romana and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G}, issn = {1546-1696}, journal = {Nature Biotechnology}, pages = {1051--1064}, publisher = {Springer Nature}, title = {{Imaging brain tissue architecture across millimeter to nanometer scales}}, doi = {10.1038/s41587-023-01911-8}, volume = {42}, year = {2024}, } @article{18603, abstract = {It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.}, author = {Kim, Olena and Okamoto, Yuji and Kaufmann, Walter and Brose, Nils and Shigemoto, Ryuichi and Jonas, Peter M}, issn = {1545-7885}, journal = {PLoS Biology}, number = {11}, publisher = {Public Library of Science}, title = {{Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons}}, doi = {10.1371/journal.pbio.3002879}, volume = {22}, year = {2024}, } @misc{18296, abstract = {It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the murine hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse.}, author = {Kim, Olena}, keywords = {Hippocampal mossy fiber synapses, short-term potentiation, long-term potentiation, presynaptic plasticity, electron microscopy, freeze-fracture replica labeling, paired recordings, forskolin, cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), neuromodulation, synaptic vesicle pools, presynaptic Ca2+ channels, Munc13, docking, priming, active zone}, publisher = {Institute of Science and Technology Austria}, title = {{Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons}}, doi = {10.15479/AT:ISTA:18296}, 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 of the United States of America}, number = {8}, publisher = {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}, number = {5}, pages = {755--771.e9}, publisher = {Elsevier}, title = {{Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse}}, doi = {10.1016/j.neuron.2023.12.002}, volume = {112}, 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}, } @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{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{13126, abstract = {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.}, author = {Danzl, Johann G}, publisher = {Institute of Science and Technology Austria}, title = {{Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales"}}, doi = {10.15479/AT:ISTA:13126}, year = {2023}, }