TY - JOUR AB - The superior colliculus (SC) in the mammalian midbrain is essential for multisensory integration and is composed of a rich diversity of excitatory and inhibitory neurons and glia. However, the developmental principles directing the generation of SC cell-type diversity are not understood. Here, we pursued systematic cell lineage tracing in silico and in vivo, preserving full spatial information, using genetic mosaic analysis with double markers (MADM)-based clonal analysis with single-cell sequencing (MADM-CloneSeq). The analysis of clonally related cell lineages revealed that radial glial progenitors (RGPs) in SC are exceptionally multipotent. Individual resident RGPs have the capacity to produce all excitatory and inhibitory SC neuron types, even at the stage of terminal division. While individual clonal units show no pre-defined cellular composition, the establishment of appropriate relative proportions of distinct neuronal types occurs in a PTEN-dependent manner. Collectively, our findings provide an inaugural framework at the single-RGP/-cell level of the mammalian SC ontogeny. AU - Cheung, Giselle T AU - Pauler, Florian AU - Koppensteiner, Peter AU - Krausgruber, Thomas AU - Streicher, Carmen AU - Schrammel, Martin AU - Özgen, Natalie Y AU - Ivec, Alexis AU - Bock, Christoph AU - Shigemoto, Ryuichi AU - Hippenmeyer, Simon ID - 12875 IS - 2 JF - Neuron SN - 0896-6273 TI - Multipotent progenitors instruct ontogeny of the superior colliculus VL - 112 ER - TY - JOUR AB - GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles (SVs) at the presynaptic active zone (AZ). Strikingly, the depressing phasic release exhibits looser coupling distance than the tonic release. Furthermore, the tonic and phasic release are selectively affected by deletion of synaptoporin (SPO) and Ca 2+ -dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. The cytosolic protein CAPS2 showed a SV-associated distribution similar to the vesicular transmembrane protein SPO, and they were colocalized in the same terminals. We developed the “Flash and Freeze-fracture” method, and revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to persistency of the RRP increase. Thus, we identified structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals. AU - Koppensteiner, Peter AU - Bhandari, Pradeep AU - Önal, Hüseyin C AU - Borges Merjane, Carolina AU - Le Monnier, Elodie AU - Roy, Utsa AU - Nakamura, Yukihiro AU - Sadakata, Tetsushi AU - Sanbo, Makoto AU - Hirabayashi, Masumi AU - Rhee, JeongSeop AU - Brose, Nils AU - Jonas, Peter M AU - Shigemoto, Ryuichi ID - 15084 IS - 8 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - GABAB receptors induce phasic release from medial habenula terminals through activity-dependent recruitment of release-ready vesicles VL - 121 ER - TY - JOUR AB - The coupling between Ca2+ channels and release sensors is a key factor defining the signaling properties of a synapse. However, the coupling nanotopography at many synapses remains unknown, and it is unclear how it changes during development. To address these questions, we examined coupling at the cerebellar inhibitory basket cell (BC)-Purkinje cell (PC) synapse. Biophysical analysis of transmission by paired recording and intracellular pipette perfusion revealed that the effects of exogenous Ca2+ chelators decreased during development, despite constant reliance of release on P/Q-type Ca2+ channels. Structural analysis by freeze-fracture replica labeling (FRL) and transmission electron microscopy (EM) indicated that presynaptic P/Q-type Ca2+ channels formed nanoclusters throughout development, whereas docked vesicles were only clustered at later developmental stages. Modeling suggested a developmental transformation from a more random to a more clustered coupling nanotopography. Thus, presynaptic signaling developmentally approaches a point-to-point configuration, optimizing speed, reliability, and energy efficiency of synaptic transmission. AU - Chen, JingJing AU - Kaufmann, Walter AU - Chen, Chong AU - Arai, Itaru AU - Kim, Olena AU - Shigemoto, Ryuichi AU - Jonas, Peter M ID - 14843 JF - Neuron SN - 0896-6273 TI - Developmental transformation of Ca2+ channel-vesicle nanotopography at a central GABAergic synapse ER - TY - THES AB - Understanding the mechanisms of learning and memory formation has always been one of the main goals in neuroscience. Already Pavlov (1927) in his early days has used his classic conditioning experiments to study the neural mechanisms governing behavioral adaptation. What was not known back then was that the part of the brain that is largely responsible for this type of associative learning is the cerebellum. Since then, plenty of theories on cerebellar learning have emerged. Despite their differences, one thing they all have in common is that learning relies on synaptic and intrinsic plasticity. The goal of my PhD project was to unravel the molecular mechanisms underlying synaptic plasticity in two synapses that have been shown to be implicated in motor learning, in an effort to understand how learning and memory formation are processed in the cerebellum. One of the earliest and most well-known cerebellar theories postulates that motor learning largely depends on long-term depression at the parallel fiber-Purkinje cell (PC-PC) synapse. However, the discovery of other types of plasticity in the cerebellar circuitry, like long-term potentiation (LTP) at the PC-PC synapse, potentiation of molecular layer interneurons (MLIs), and plasticity transfer from the cortex to the cerebellar/ vestibular nuclei has increased the popularity of the idea that multiple sites of plasticity might be involved in learning. Still a lot remains unknown about the molecular mechanisms responsible for these types of plasticity and whether they occur during physiological learning. In the first part of this thesis we have analyzed the variation and nanodistribution of voltagegated calcium channels (VGCCs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid type glutamate receptors (AMPARs) on the parallel fiber-Purkinje cell synapse after vestibuloocular reflex phase reversal adaptation, a behavior that has been suggested to rely on PF-PC LTP. We have found that on the last day of adaptation there is no learning trace in form of VGCCs nor AMPARs variation at the PF-PC synapse, but instead a decrease in the number of PF-PC synapses. These data seem to support the view that learning is only stored in the cerebellar cortex in an initial learning phase, being transferred later to the vestibular nuclei. Next, we have studied the role of MLIs in motor learning using a relatively simple and well characterized behavioral paradigm – horizontal optokinetic reflex (HOKR) adaptation. We have found behavior-induced MLI potentiation in form of release probability increase that could be explained by the increase of VGCCs at the presynaptic side. Our results strengthen the idea of distributed cerebellar plasticity contributing to learning and provide a novel mechanism for release probability increase. AU - Alcarva, Catarina ID - 12809 SN - 2663 - 337X TI - Plasticity in the cerebellum: What molecular mechanisms are behind physiological learning ER - TY - JOUR AB - Junctions between the endoplasmic reticulum (ER) and the plasma membrane (PM) are specialized membrane contacts ubiquitous in eukaryotic cells. Concentration of intracellular signaling machinery near ER-PM junctions allows these domains to serve critical roles in lipid and Ca2+ signaling and homeostasis. Subcellular compartmentalization of protein kinase A (PKA) signaling also regulates essential cellular functions, however, no specific association between PKA and ER-PM junctional domains is known. Here, we show that in brain neurons type I PKA is directed to Kv2.1 channel-dependent ER-PM junctional domains via SPHKAP, a type I PKA-specific anchoring protein. SPHKAP association with type I PKA regulatory subunit RI and ER-resident VAP proteins results in the concentration of type I PKA between stacked ER cisternae associated with ER-PM junctions. This ER-associated PKA signalosome enables reciprocal regulation between PKA and Ca2+ signaling machinery to support Ca2+ influx and excitation-transcription coupling. These data reveal that neuronal ER-PM junctions support a receptor-independent form of PKA signaling driven by membrane depolarization and intracellular Ca2+, allowing conversion of information encoded in electrical signals into biochemical changes universally recognized throughout the cell. AU - Vierra, Nicholas C. AU - Ribeiro-Silva, Luisa AU - Kirmiz, Michael AU - Van Der List, Deborah AU - Bhandari, Pradeep AU - Mack, Olivia A. AU - Carroll, James AU - Le Monnier, Elodie AU - Aicher, Sue A. AU - Shigemoto, Ryuichi AU - Trimmer, James S. ID - 14253 JF - Nature Communications TI - Neuronal ER-plasma membrane junctions couple excitation to Ca2+-activated PKA signaling VL - 14 ER - TY - JOUR AB - Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P2 in cryo-fixed brain. We demonstrate that PI(4,5)P2 forms tiny clusters with a mean size of ∼1000 nm2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P2 with CaV2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P2 in neurons. AU - Eguchi, Kohgaku AU - Le Monnier, Elodie AU - Shigemoto, Ryuichi ID - 13202 IS - 23 JF - The Journal of Neuroscience SN - 0270-6474 TI - Nanoscale phosphoinositide distribution on cell membranes of mouse cerebellar neurons VL - 43 ER - TY - DATA AB - 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 - Danzl, Johann G ID - 13126 TI - Research data for the publication "Imaging brain tissue architecture across millimeter to nanometer scales" 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 - DATA AB - GABAB receptor (GBR) activation inhibits neurotransmitter release in axon terminals in the brain, except in medial habenula (MHb) terminals, which show robust potentiation. However, mechanisms underlying this enigmatic potentiation remain elusive. Here, we report that GBR activation on MHb terminals induces an activity-dependent transition from a facilitating, tonic to a depressing, phasic neurotransmitter release mode. This transition is accompanied by a 4.1-fold increase in readily releasable vesicle pool (RRP) size and a 3.5-fold increase of docked synaptic vesicles at the presynaptic active zone (AZ). Strikingly, tonic and phasic release exhibit distinct coupling distances and are selectively affected by deletion of synaptoporin (SPO) and Ca2+-dependent activator protein for secretion 2 (CAPS2), respectively. SPO modulates augmentation, the short-term plasticity associated with tonic release, and CAPS2 retains the increased RRP for initial responses in phasic response trains. Double pre-embedding immunolabeling confirmed the co-localization of CAPS2 and SPO inside the same terminal. The cytosolic protein CAPS2 showed a synaptic vesicle (SV)-associated distribution similar to the vesicular transmembrane protein SPO. A newly developed “Flash and Freeze-fracture” method revealed the release of SPO-associated vesicles in both tonic and phasic modes and activity-dependent recruitment of CAPS2 to the AZ during phasic release, which lasted several minutes. Overall, these results indicate that GBR activation translocates CAPS2 to the AZ along with the fusion of CAPS2-associated SVs, contributing to a persistent RRP increase. Thus, we discovered structural and molecular mechanisms underlying tonic and phasic neurotransmitter release and their transition by GBR activation in MHb terminals. AU - Shigemoto, Ryuichi ID - 13173 KW - medial habenula KW - GABAB receptor KW - vesicle release KW - Flash and Freeze KW - Flash and Freeze-fracture TI - Transition from tonic to phasic neurotransmitter release by presynaptic GABAB receptor activation in medial habenula terminals ER - TY - JOUR AB - Upon the arrival of action potentials at nerve terminals, neurotransmitters are released from synaptic vesicles (SVs) by exocytosis. CaV2.1, 2.2, and 2.3 are the major subunits of the voltage-gated calcium channel (VGCC) responsible for increasing intraterminal calcium levels and triggering SV exocytosis in the central nervous system (CNS) synapses. The two-dimensional analysis of CaV2 distributions using sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) has revealed their numbers, densities, and nanoscale clustering patterns in individual presynaptic active zones. The variation in these properties affects the coupling of VGCCs with calcium sensors on SVs, synaptic efficacy, and temporal precision of transmission. In this study, we summarize how the morphological parameters of CaV2 distribution obtained using SDS-FRL differ depending on the different types of synapses and could correspond to functional properties in synaptic transmission. AU - Eguchi, Kohgaku AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Le Monnier, Elodie AU - Shigemoto, Ryuichi ID - 10890 JF - Frontiers in Neuroanatomy TI - The number and distinct clustering patterns of voltage-gated Calcium channels in nerve terminals VL - 16 ER -