@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{14683, abstract = {Mosaic analysis with double markers (MADM) technology enables the generation of genetic mosaic tissue in mice and high-resolution phenotyping at the individual cell level. Here, we present a protocol for isolating MADM-labeled cells with high yield for downstream molecular analyses using fluorescence-activated cell sorting (FACS). We describe steps for generating MADM-labeled mice, perfusion, single-cell suspension, and debris removal. We then detail procedures for cell sorting by FACS and downstream analysis. This protocol is suitable for embryonic to adult mice. For complete details on the use and execution of this protocol, please refer to Contreras et al. (2021).1}, author = {Amberg, Nicole and Cheung, Giselle T and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, keywords = {General Immunology and Microbiology, General Biochemistry, Genetics and Molecular Biology, General Neuroscience}, number = {1}, publisher = {Elsevier}, title = {{Protocol for sorting cells from mouse brains labeled with mosaic analysis with double markers by flow cytometry}}, doi = {10.1016/j.xpro.2023.102771}, volume = {5}, year = {2024}, } @article{17187, abstract = {The generation of diverse cell types during development is fundamental to brain functions. We outline a protocol to quantitatively assess the clonal output of individual neural progenitors using mosaic analysis with double markers (MADM) in mice. We first describe steps to acquire and reconstruct adult MADM clones in the superior colliculus. Then we detail analysis pipelines to determine clonal composition and architecture. This protocol enables the buildup of quantitative frameworks of lineage progression with precise spatial resolution in the brain. For complete details on the use and execution of this protocol, please refer to Cheung et al.1}, author = {Cheung, Giselle T and Streicher, Carmen and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {3}, publisher = {Elsevier}, title = {{Protocol for quantitative reconstruction of cell lineage using mosaic analysis with double markers in mice}}, doi = {10.1016/j.xpro.2024.103157}, volume = {5}, year = {2024}, } @article{17232, abstract = {The lineage relationship of clonally-related cells offers important insights into the ontogeny and cytoarchitecture of the brain in health and disease. Here, we provide a protocol to concurrently assess cell lineage relationship and cell-type identity among clonally-related cells in situ. We first describe the preparation and screening of acute brain slices containing clonally-related cells labeled using mosaic analysis with double markers (MADM). We then outline steps to collect RNA from individual cells for downstream applications and cell-type identification using RNA sequencing. For complete details on the use and execution of this protocol, please refer to Cheung et al. 1}, author = {Cheung, Giselle T and Pauler, Florian and Koppensteiner, Peter and Hippenmeyer, Simon}, issn = {2666-1667}, journal = {STAR Protocols}, number = {3}, publisher = {Elsevier}, title = {{Protocol for mapping cell lineage and cell-type identity of clonally-related cells in situ using MADM-CloneSeq}}, doi = {10.1016/j.xpro.2024.103168}, volume = {5}, year = {2024}, } @inbook{17425, abstract = {Mosaic Analysis with Double Markers (MADM) is a powerful genetic method typically used for lineage tracing and to disentangle cell autonomous and tissue-wide roles of candidate genes with single cell resolution. Given the relatively sparse labeling, depending on which of the 19 MADM chromosomes one chooses, the MADM approach represents the perfect opportunity for cell morphology analysis. Various MADM studies include reports of morphological anomalies and phenotypes in the central nervous system (CNS). MADM for any candidate gene can easily incorporate morphological analysis within the experimental workflow. Here, we describe the methods of morphological cell analysis which we developed in the course of diverse recent MADM studies. This chapter will specifically focus on methods to quantify aspects of the morphology of neurons and astrocytes within the CNS, but these methods can broadly be applied to any MADM-labeled cells throughout the entire organism. We will cover two analyses—soma volume and dendrite characterization—of physical characteristics of pyramidal neurons in the somatosensory cortex, and two analyses—volume and Sholl analysis—of astrocyte morphology.}, author = {Miranda, Osvaldo and Cheung, Giselle T and Hippenmeyer, Simon}, booktitle = {Neuronal Morphogenesis}, editor = {Toyooka, Kazuhito}, isbn = {9781071639689}, issn = {1940-6029}, pages = {283--299}, publisher = {Springer Nature}, title = {{Morphological Analysis of Neurons and Glia Using Mosaic Analysis with Double Markers}}, doi = {10.1007/978-1-0716-3969-6_19}, volume = {2831}, year = {2024}, } @unpublished{18688, abstract = {The human brain has remarkable computational power. It generates sophisticated behavioral sequences, stores engrams over an individual’s lifetime, and produces higher cognitive functions up to the level of consciousness. However, so little of our neuroscience knowledge covers the human brain, and it remains unknown whether this organ is truly unique, or is a scaled version of the extensively studied rodent brain. To address this fundamental question, we determined the cellular, synaptic, and connectivity rules of the hippocampal CA3 recurrent circuit using multicellular patch clamp-recording. This circuit is the largest autoassociative network in the brain, and plays a key role in memory and higher-order computations such as pattern separation and pattern completion. We demonstrate that human hippocampal CA3 employs sparse connectivity, in stark contrast to neocortical recurrent networks. Connectivity sparsifies from rodents to humans, providing a circuit architecture that maximizes associational power. Unitary synaptic events at human CA3–CA3 synapses showed both distinct species-specific and circuit-dependent properties, with high reliability, unique amplitude precision, and long integration times. We also identify differential scaling rules between hippocampal pathways from rodents to humans, with a moderate increase in the convergence of CA3 inputs per cell, but a marked increase in human mossy fiber innervation. Anatomically guided full-scale modeling suggests that the human brain’s sparse connectivity, expanded neuronal number, and reliable synaptic signaling combine to enhance the associative memory storage capacity of CA3. Together, our results reveal unique rules of connectivity and synaptic signaling in the human hippocampus, demonstrating the absolute necessity of human brain research and beginning to unravel the remarkable performance of our autoassociative memory circuits.}, author = {Watson, Jake F. and Vargas-Barroso, Victor and Morse-Mora, Rebecca J. and Navas-Olive, Andrea and Tavakoli, Mojtaba and Danzl, Johann G and Tomschik, Matthias and Rössler, Karl and Jonas, Peter M}, booktitle = {bioRxiv}, title = {{Human hippocampal CA3 uses specific functional connectivity rules for efficient associative memory}}, doi = {10.1101/2024.05.02.592169}, 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}, } @phdthesis{18674, abstract = {Mapping the complex and dense arrangement of cells and their connectivity in brain tissue requires volumetric imaging at nanoscale spatial resolution. While light microscopy excels at visualizing specific molecules and individual cells, achieving dense, synapse-level circuit reconstruction has not been possible with any light microscopy technique. Thus, the goal of my work was to develop image and data analysis pipelines for brain tissue visualization and reconstruction with light microscopy. To achieve dense circuit reconstruction with single-synapse resolution, I developed both conventional and deep-learning-based synapse detection algorithms, as well as connectivity analysis pipelines that integrate synapse detection with volumetric segmentation of brain tissue.}, author = {Lyudchik, Julia}, isbn = { 978-3-99078-051-0}, issn = {2663-337X}, pages = {217}, publisher = {Institute of Science and Technology Austria}, title = {{Image analysis for brain tissue reconstruction with super-resolution light microscopy}}, doi = {10.15479/at:ista:18674}, year = {2024}, } @article{18481, abstract = {A tight regulation of morphogen production is key for morphogen gradient formation and thereby for reproducible and organised organ development. Although many genetic interactions involved in the establishment of morphogen production domains are known, the biophysical mechanisms of morphogen source formation are poorly understood. Here we addressed this by focusing on the morphogen Sonic hedgehog (Shh) in the vertebrate neural tube. Shh is produced by the adjacently located notochord and by the floor plate of the neural tube. Using a data-constrained computational screen, we identified different possible mechanisms by which floor plate formation can occur, only one of which is consistent with experimental data. In this mechanism, the floor plate is established rapidly in response to Shh from the notochord and the dynamics of regulatory interactions within the neural tube. In this process, uniform activators and Shh-dependent repressors are key for establishing the floor plate size. Subsequently, the floor plate becomes insensitive to Shh and increases in size due to tissue growth, leading to scaling of the floor plate with neural tube size. In turn, this results in scaling of the Shh amplitude with tissue growth. Thus, this mechanism ensures a separation of time scales in floor plate formation, so that the floor plate domain becomes growth-dependent after an initial rapid establishment phase. Our study raises the possibility that the time scale separation between specification and growth might be a common strategy for scaling the morphogen gradient amplitude in growing organs. The model that we developed provides a new opportunity for quantitative studies of morphogen source formation in growing tissues.}, author = {Ho, Richard D.J.G. and Kishi, Kasumi and Majka, Maciej and Kicheva, Anna and Zagórski, Marcin P}, issn = {1553-7358}, journal = {PLoS Computational Biology}, publisher = {Public Library of Science}, title = {{Dynamics of morphogen source formation in a growing tissue}}, doi = {10.1371/journal.pcbi.1012508}, volume = {20}, year = {2024}, } @article{18902, author = {Zagorski, Marcin and Brandenberg, Nathalie and Lutolf, Matthias and Tkačik, Gašper and Bollenbach, Mark Tobias and Briscoe, James and Kicheva, Anna}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Assessing the precision of morphogen gradients in neural tube development}}, doi = {10.1038/s41467-024-45148-8}, volume = {15}, year = {2024}, } @phdthesis{17133, abstract = {An ideal quantum computer relies on qubits capable of performing fast gate operations and maintaining strong interconnections while preserving their quantum coherence. Since the inception of experimental eforts toward building a quantum computer, the community has faced challenges in engineering such a system. Among the various methods of implementing a quantum computer, superconducting qubits have shown fast gates close to tens of nanoseconds, with the state-of-the-art reaching a coherence of a few milliseconds. However, achieving simultaneously long lifetimes with fast qubit operations poses an inherent paradox. Qubits with high coherence require isolation from the environment, while fast operation necessitates strong coupling of the qubit. This thesis approaches this issue by proposing the idea of engineering superconducting qubits capable of transitioning between operating in a protected regime, where the qubit is completely isolated from the environment, and coupling to the communication channels as needed. In this direction, we use the geometric superinductor to scan the parameter space of rf-SQUID devices, searching for a regime where we can take the qubit protection to its extreme. This leads us to the inductively shunted transmon (IST) regime, characterized by EJ /EC ≫ 1 and EJ /EL ≫ 1, where the circuit potential exhibits a double well with a large barrier separating the local ground states of each quantum well. In this regime, although it is anticipated that the two quantum wells would be isolated from each other, we observe single fuxon tunneling between them. The interplay of the cavity photons and the fuxon transition forms a rich physical system, containing resonance conditions that allow the preparation of the fuxon ground or excited states. This enables us to study the relaxation rate of such transition and show that it can be as large as 3.6 hours. Dynamically controlling the barrier height between the two quantum wells allows for controllable coupling, which scales exponentially, for a qubit encoded in two fuxon states. The 0-π qubit is one of the very few known superconducting circuit types that ofers exponential protection from both relaxation and dephasing simultaneously. However, this qubit is not exempt from the fact that such protection comes at the expense of complex readout and control. In this thesis, we propose a way to controllably break the circuit symmetry, the key reason for the protection, to momentarily restore the ability to control and manipulate the qubit. An asymmetry in capacitances and inductances in the 0-π circuit is detrimental since they lead to coupling of the protected state to the thermally occupied parasitic mode of the circuit. However, here we try to exploit a controlled asymmetry in Josephson energies and show that this can be used as a tunable coupler between the protected states. In the future, this should allow to perform gate operations by dynamically controlling the asymmetry instead of driving the protected transition with microwave pulses. Therefore, we believe that the proposed method can make the use of protected qubits more practical in experimental realizations of quantum computing.}, author = {Hassani, Farid}, isbn = {978-3-99078-040-4}, issn = {2663-337X}, keywords = {Quantum information, Qubits, Superconducting devices}, pages = {161}, publisher = {Institute of Science and Technology Austria}, title = {{Superconducting qubits capable of dynamic switching between protected and high-speed control regimes}}, doi = {10.15479/at:ista:17133}, year = {2024}, } @article{15362, abstract = {Constitutional heterozygous pathogenic variants in the exonuclease domain of POLE and POLD1, which affect the proofreading activity of the corresponding polymerases, cause a cancer predisposition syndrome characterized by increased risk of gastrointestinal polyposis, colorectal cancer, endometrial cancer and other tumor types. The generally accepted explanation for the connection between the disruption of the proofreading activity of polymerases epsilon and delta and cancer development is through an increase in the somatic mutation rate. Here we studied an extended family with multiple members heterozygous for the pathogenic POLD1 variant c.1421T>C p.(Leu474Pro), which segregates with the polyposis and cancer phenotypes. Through the analysis of mutational patterns of patient-derived fibroblasts colonies and de novo mutations obtained by parent-offspring comparisons, we concluded that heterozygous POLD1 L474P just subtly increases the somatic and germline mutation burden. In contrast, tumors developed in individuals with a heterozygous mutation in the exonuclease domain of POLD1, including L474P, have an extremely high mutation rate (>100 mut/Mb) associated with signature SBS10d. We solved this contradiction through the observation that tumorigenesis involves somatic inactivation of the wildtype POLD1 allele. These results imply that exonuclease deficiency of polymerase delta has a recessive effect on mutation rate.}, author = {Andrianova, Maria A. and Seplyarskiy, Vladimir B. and Terradas, Mariona and Sánchez-Heras, Ana Beatriz and Mur, Pilar and Soto, José Luis and Aiza, Gemma and Borràs, Emma and Kondrashov, Fyodor and Kondrashov, Alexey S. and Bazykin, Georgii A. and Valle, Laura}, issn = {1476-5438}, journal = {European Journal of Human Genetics}, pages = {837--845}, publisher = {Springer Nature}, title = {{Discovery of recessive effect of human polymerase δ proofreading deficiency through mutational analysis of POLD1-mutated normal and cancer cells}}, doi = {10.1038/s41431-024-01598-8}, volume = {32}, year = {2024}, } @phdthesis{18135, abstract = {This thesis consists of two separate parts. In the first part we consider a dilute Fermi gas interacting through a repulsive interaction in dimensions $d=1,2,3$. Our focus is mostly on the physically most relevant dimension $d=3$ and the setting of a spin-polarized (equivalently spinless) gas, where the Pauli exclusion principle plays a key role. We show that, at zero temperature, the ground state energy density of the interacting spin-polarized gas differs (to leading order) from that of the free (i.e. non-interacting) gas by a term of order $a_p^d\rho^{2+2/d}$ with $a_p$ the $p$-wave scattering length of the repulsive interaction and $\rho$ the density. Further, we extend this to positive temperature and show that the pressure of an interacting spin-polarized gas differs from that of the free gas by a now temperature dependent term, again of order $a_p^d\rho^{2+2/d}$. Lastly, we consider the setting of a spin-$\frac{1}{2}$ Fermi gas in $d=3$ dimensions and show that here, as an upper bound, the ground state energy density differs from that of the free system by a term of order $a_s \rho^2$ with an error smaller than $a_s \rho^2 (a_s\rho^{1/3})^{1-\eps}$ for any $\eps > 0$, where $a_s$ is the $s$-wave scattering length of the repulsive interaction. These asymptotic formulas complement the similar formulas in the literature for the dilute Bose and spin-$\frac{1}{2}$ Fermi gas, where the ground state energies or pressures differ from that of the corresponding free systems by a term of order $a_s \rho^2$ in dimension $d=3$. In the spin-polarized setting, the corrections, of order $a_p^3\rho^{8/3}$ in dimension $d=3$, are thus much smaller and requires a more delicate analysis. In the second part of the thesis we consider the Bardeen--Cooper--Schrieffer (BCS) theory of superconductivity and in particular its associated critical temperature and energy gap. We prove that the ratio of the zero-temperature energy gap and critical temperature $\Xi(T=0)/T_c$ approaches a universal constant $\pi e^{-\gamma}\approx 1.76$ in both the limit of high density in dimension $d=3$ and in the limit of weak coupling in dimensions $d=1,2$. This complements the proofs in the literature of this universal behaviour in the limit of weak coupling or low density in dimension $d=3$. Secondly, we prove that the ratio of the energy gap at positive temperature and critical temperature $\Xi(T)/T_c$ approaches a universal function of the relative temperature $T/T_c$ in the limit of weak coupling in dimensions $d=1,2,3$.}, author = {Lauritsen, Asbjørn Bækgaard}, isbn = {978-3-99078-042-8}, issn = {2663-337X}, pages = {353}, publisher = {Institute of Science and Technology Austria}, title = {{Energies of dilute Fermi gases and universalities in BCS theory}}, doi = {10.15479/at:ista:18135}, year = {2024}, } @article{14931, abstract = {We prove an upper bound on the ground state energy of the dilute spin-polarized Fermi gas capturing the leading correction to the kinetic energy resulting from repulsive interactions. One of the main ingredients in the proof is a rigorous implementation of the fermionic cluster expansion of Gaudin et al. (1971) [15].}, author = {Lauritsen, Asbjørn Bækgaard and Seiringer, Robert}, issn = {1096-0783}, journal = {Journal of Functional Analysis}, number = {7}, publisher = {Elsevier}, title = {{Ground state energy of the dilute spin-polarized Fermi gas: Upper bound via cluster expansion}}, doi = {10.1016/j.jfa.2024.110320}, volume = {286}, year = {2024}, } @inproceedings{15012, abstract = {We solve a problem of Dujmović and Wood (2007) by showing that a complete convex geometric graph on n vertices cannot be decomposed into fewer than n-1 star-forests, each consisting of noncrossing edges. This bound is clearly tight. We also discuss similar questions for abstract graphs.}, author = {Pach, János and Saghafian, Morteza and Schnider, Patrick}, booktitle = {31st International Symposium on Graph Drawing and Network Visualization}, isbn = {9783031492716}, issn = {1611-3349}, location = {Isola delle Femmine, Palermo, Italy}, pages = {339--346}, publisher = {Springer Nature}, title = {{Decomposition of geometric graphs into star-forests}}, doi = {10.1007/978-3-031-49272-3_23}, volume = {14465}, year = {2024}, } @article{12738, abstract = {We study turn-based stochastic zero-sum games with lexicographic preferences over objectives. Stochastic games are standard models in control, verification, and synthesis of stochastic reactive systems that exhibit both randomness as well as controllable and adversarial non-determinism. Lexicographic order allows one to consider multiple objectives with a strict preference order. To the best of our knowledge, stochastic games with lexicographic objectives have not been studied before. For a mixture of reachability and safety objectives, we show that deterministic lexicographically optimal strategies exist and memory is only required to remember the already satisfied and violated objectives. For a constant number of objectives, we show that the relevant decision problem is in NP∩coNP, matching the current known bound for single objectives; and in general the decision problem is PSPACE-hard and can be solved in NEXPTIME∩coNEXPTIME. We present an algorithm that computes the lexicographically optimal strategies via a reduction to the computation of optimal strategies in a sequence of single-objectives games. For omega-regular objectives, we restrict our analysis to one-player games, also known as Markov decision processes. We show that lexicographically optimal strategies exist and need either randomization or finite memory. We present an algorithm that solves the relevant decision problem in polynomial time. We have implemented our algorithms and report experimental results on various case studies.}, author = {Chatterjee, Krishnendu and Katoen, Joost P and Mohr, Stefanie and Weininger, Maximilian and Winkler, Tobias}, issn = {1572-8102}, journal = {Formal Methods in System Design}, pages = {40--80}, publisher = {Springer Nature}, title = {{Stochastic games with lexicographic objectives}}, doi = {10.1007/s10703-023-00411-4}, volume = {63}, year = {2024}, } @article{14793, abstract = {Superconductor/semiconductor hybrid devices have attracted increasing interest in the past years. Superconducting electronics aims to complement semiconductor technology, while hybrid architectures are at the forefront of new ideas such as topological superconductivity and protected qubits. In this work, we engineer the induced superconductivity in two-dimensional germanium hole gas by varying the distance between the quantum well and the aluminum. We demonstrate a hard superconducting gap and realize an electrically and flux tunable superconducting diode using a superconducting quantum interference device (SQUID). This allows to tune the current phase relation (CPR), to a regime where single Cooper pair tunneling is suppressed, creating a sin(2y) CPR. Shapiro experiments complement this interpretation and the microwave drive allows to create a diode with ≈ 100% efficiency. The reported results open up the path towards integration of spin qubit devices, microwave resonators and (protected) superconducting qubits on the same silicon technology compatible platform.}, author = {Valentini, Marco and Sagi, Oliver and Baghumyan, Levon and de Gijsel, Thijs and Jung, Jason and Calcaterra, Stefano and Ballabio, Andrea and Aguilera Servin, Juan L and Aggarwal, Kushagra and Janik, Marian and Adletzberger, Thomas and Seoane Souto, Rubén and Leijnse, Martin and Danon, Jeroen and Schrade, Constantin and Bakkers, Erik and Chrastina, Daniel and Isella, Giovanni and Katsaros, Georgios}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Parity-conserving Cooper-pair transport and ideal superconducting diode in planar germanium}}, doi = {10.1038/s41467-023-44114-0}, volume = {15}, 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{15401, abstract = {Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H2O-based culture medium doped with deuterated cell lysate allow scientists to overcome this “reprotonation bottleneck” while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments.}, author = {Napoli, Federico and Guan, Jia-Ying and Arnaud, Charles-Adrien and Macek, Pavel and Fraga, Hugo and Breyton, Cécile and Schanda, Paul}, issn = {2699-0016}, journal = {Magnetic Resonance}, number = {1}, pages = {33--49}, publisher = {Copernicus Publications}, title = {{Deuteration of proteins boosted by cell lysates: High-resolution amide and Ha magic-angle-spinning (MAS) NMR without the reprotonation bottleneck}}, doi = {10.5194/mr-5-33-2024}, volume = {5}, year = {2024}, }