@article{18879, abstract = {Our brain has remarkable computational power, generating sophisticated behaviors, storing memories over an individual’s lifetime, and producing higher cognitive functions. However, little of our neuroscience knowledge covers the human brain. Is this organ truly unique, or is it a scaled version of the extensively studied rodent brain? Combining multicellular patch-clamp recording with expansion-based superresolution microscopy and full-scale modeling, we determined the cellular and microcircuit properties of the human hippocampal CA3 region, a fundamental circuit for memory storage. In contrast to neocortical networks, human hippocampal CA3 displayed sparse connectivity, providing a circuit architecture that maximizes associational power. Human synapses showed unique reliability, high precision, and long integration times, exhibiting both species- and circuit-specific properties. Together with expanded neuronal numbers, these circuit characteristics greatly enhanced the memory storage capacity of CA3. Our results reveal distinct microcircuit properties of the human hippocampus and begin to unravel the inner workings of our most complex organ. }, author = {Watson, Jake and Vargas Barroso, Victor M and Morse, Rebecca and Navas Olivé, Andrea C and Tavakoli, Mojtaba and Danzl, Johann G and Tomschik, Matthias and Rössler, Karl and Jonas, Peter M}, issn = {1097-4172}, journal = {Cell}, number = {2}, pages = {501--514.e18}, publisher = {Elsevier}, title = {{Human hippocampal CA3 uses specific functional connectivity rules for efficient associative memory}}, doi = {10.1016/j.cell.2024.11.022}, volume = {188}, year = {2025}, } @inbook{18058, abstract = {DNA cloning is a core technique in biomedical and biotechnological research and is used to assemble and modify DNA fragments at will. While DNA cloning has traditionally relied on restriction enzymes, recent homology-based methods offer improved protocols together with seamless and directional assembly of desired products, overcoming the main disadvantages of restriction enzyme DNA cloning. This chapter provides a historical perspective on DNA cloning, presents a detailed discussion on state-of-the-art in vitro and in vivo homology-based methodologies, covering the basics of how to perform all major plasmid modifications (sub-cloning, site-directed mutagenesis, insertions, and deletions), and gives examples of how to apply these techniques for complex DNA cloning projects.}, author = {Watson, Jake and Arroyo-Urea, Sandra and García-Nafría, Javier}, booktitle = {Handbook of Molecular Biotechnology}, editor = {Liu, Dongyou}, pages = {66--72}, publisher = {CRC Press}, title = {{DNA Cloning}}, doi = {10.1201/9781003055211-8}, year = {2024}, } @article{15117, abstract = {The hippocampal mossy fiber synapse, formed between axons of dentate gyrus granule cells and dendrites of CA3 pyramidal neurons, is a key synapse in the trisynaptic circuitry of the hippocampus. Because of its comparatively large size, this synapse is accessible to direct presynaptic recording, allowing a rigorous investigation of the biophysical mechanisms of synaptic transmission and plasticity. Furthermore, because of its placement in the very center of the hippocampal memory circuit, this synapse seems to be critically involved in several higher network functions, such as learning, memory, pattern separation, and pattern completion. Recent work based on new technologies in both nanoanatomy and nanophysiology, including presynaptic patch-clamp recording, paired recording, super-resolution light microscopy, and freeze-fracture and “flash-and-freeze” electron microscopy, has provided new insights into the structure, biophysics, and network function of this intriguing synapse. This brings us one step closer to answering a fundamental question in neuroscience: how basic synaptic properties shape higher network computations.}, author = {Vandael, David H and Jonas, Peter M}, issn = {1095-9203}, journal = {Science}, number = {6687}, pages = {eadg6757}, publisher = {AAAS}, title = {{Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal}}, doi = {10.1126/science.adg6757}, volume = {383}, year = {2024}, } @article{15379, abstract = {Long-term potentiation (LTP) of excitatory synapses is a leading model to explain the concept of information storage in the brain. Multiple mechanisms contribute to LTP, but central amongst them is an increased sensitivity of the postsynaptic membrane to neurotransmitter release. This sensitivity is predominantly determined by the abundance and localization of AMPA-type glutamate receptors (AMPARs). A combination of AMPAR structural data, super-resolution imaging of excitatory synapses, and an abundance of electrophysiological studies are providing an ever-clearer picture of how AMPARs are recruited and organized at synaptic junctions. Here, we review the latest insights into this process, and discuss how both cytoplasmic and extracellular receptor elements cooperate to tune the AMPAR response at the hippocampal CA1 synapse.}, author = {Stockwell, Imogen and Watson, Jake and Greger, Ingo H.}, issn = {1521-1878}, journal = {BioEssays}, number = {7}, publisher = {Wiley}, title = {{Tuning synaptic strength by regulation of AMPA glutamate receptor localization}}, doi = {10.1002/bies.202400006}, volume = {46}, year = {2024}, } @article{17122, abstract = {Background: Motor alterations and lowered physical activity are common in affective disorders. Previous research has indicated a link between depressive symptoms and declining muscle strength primarily focusing on the elderly but not younger individuals. Thus, we aimed to evaluate the relationship between mood and muscle strength in a sample of N = 73 young to middle-aged hospitalized patients (18–49 years, mean age 30.7 years) diagnosed with major depressive, bipolar and schizoaffective disorder, with a focus on moderating effects of psychopharmacotherapy. The study was carried out as a prospective observational study at a German psychiatric university hospital between September 2021 and March 2022. Methods: Employing a standardized strength circuit consisting of computerized strength training devices, we measured the maximal muscle strength (Fmax) using three repetitions maximum across four muscle regions (abdomen, arm, back, leg) at three time points (t1-t3) over four weeks accompanied by psychometric testing (MADRS, BPRS, YRMS) and blood lipid profiling in a clinical setting. For analysis of psychopharmacotherapy, medication was split into activating (AM) and inhibiting (IM) medication and dosages were normalized by the respective WHO defined daily dose. Results: While we observed a significant decrease of the MADRS score and increase of the relative total Fmax (rTFmax) in the first two weeks (t1-t2) but not later (both p < .001), we did not reveal a significant bivariate correlation between disease severity (MADRS) and muscle strength (rTFmax) at any of the timepoints. Individuals with longer disease history displayed reduced rTFmax (p = .048). IM was significantly associated with decreased rTFmax (p = .032). Regression models provide a more substantial effect of gender, age, and IM on muscle strength than the depressive episode itself (p < .001). Conclusions: The results of the study indicate that disease severity and muscle strength are not associated in young to middle-aged inpatients with affective disorders using a strength circuit as observational measurement. Future research will be needed to differentiate the effect of medication, gender, and age on muscle strength and to develop interventions for prevention of muscle weakness, especially in younger patients with chronic affective illnesses.}, author = {Ramming, Hannah and Theuerkauf, Linda and Hoos, Olaf and Lichter, Katharina and Kittel-Schneider, Sarah}, issn = {1471-244X}, journal = {BMC Psychiatry}, publisher = {Springer Nature}, title = {{The association between maximal muscle strength, disease severity and psychopharmacotherapy among young to middle-aged inpatients with affective disorders – a prospective pilot study}}, doi = {10.1186/s12888-024-05849-2}, volume = {24}, 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}, } @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}, } @phdthesis{15101, abstract = {The coupling between presynaptic Ca2+ channels and release sensors is a key factor that determines speed and efficacy of synapse transmission. At some excitatory synapses, channel–sensor coupling becomes tighter during development, and tightening is often associated with a switch in the reliance on different Ca2+ channel subtypes. However, the coupling topography at many synapses remains unknown, and it is unclear how it changes during development. To address this question, we analyzed the coupling configuration at the cerebellar basket cell (BC) to Purkinje cell (PC) synapse at different developmental stages, combining biophysical analysis, structural analysis, and modeling. Quantal analysis of BC–PC indicated that release probability decreased, while the number of functional sites increased during development. Although transmitter release persistently relied on P/Q-type Ca2+ channels in the time period postnatal day 7–23, effects of the Ca2+ chelator EGTA and BAPTA applied by intracellular pipette perfusion decreased during development, indicative of tightening of source-sensor coupling. Furthermore, presynaptic action potentials became shorter during development, suggesting reduced efficacy of Ca2+ channel activation. 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. The number of functional release sites correlated better with the AZ number early in development, but match better with the Ca2+ channel cluster number at later 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}, issn = {2663-337X}, pages = {84}, publisher = {Institute of Science and Technology Austria}, title = {{Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse}}, doi = {10.15479/at:ista:15101}, year = {2024}, } @article{12515, abstract = {Introduction: The olfactory system in most mammals is divided into several subsystems based on the anatomical locations of the neuroreceptor cells involved and the receptor families that are expressed. In addition to the main olfactory system and the vomeronasal system, a range of olfactory subsystems converge onto the transition zone located between the main olfactory bulb (MOB) and the accessory olfactory bulb (AOB), which has been termed the olfactory limbus (OL). The OL contains specialized glomeruli that receive noncanonical sensory afferences and which interact with the MOB and AOB. Little is known regarding the olfactory subsystems of mammals other than laboratory rodents. Methods: We have focused on characterizing the OL in the red fox by performing general and specific histological stainings on serial sections, using both single and double immunohistochemical and lectin-histochemical labeling techniques. Results: As a result, we have been able to determine that the OL of the red fox (Vulpes vulpes) displays an uncommonly high degree of development and complexity. Discussion: This makes this species a novel mammalian model, the study of which could improve our understanding of the noncanonical pathways involved in the processing of chemosensory cues.}, author = {Ortiz-Leal, Irene and Torres, Mateo V. and Vargas Barroso, Victor M and Fidalgo, Luis Eusebio and López-Beceiro, Ana María and Larriva-Sahd, Jorge A. and Sánchez-Quinteiro, Pablo}, issn = {1662-5129}, journal = {Frontiers in Neuroanatomy}, publisher = {Frontiers}, title = {{The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway}}, doi = {10.3389/fnana.2022.1097467}, volume = {16}, year = {2023}, } @article{12567, abstract = {Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations.}, author = {Mrestani, Achmed and Lichter, Katharina and Sirén, Anna Leena and Heckmann, Manfred and Paul, Mila M. and Pauli, Martin}, issn = {1422-0067}, journal = {International Journal of Molecular Sciences}, number = {3}, publisher = {MDPI}, title = {{Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation}}, doi = {10.3390/ijms24032128}, volume = {24}, year = {2023}, } @inbook{12720, abstract = {Here we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues.}, author = {Arroyo-Urea, Sandra and Watson, Jake and García-Nafría, Javier}, booktitle = {DNA Manipulation and Analysis}, editor = {Scarlett, Garry}, isbn = {978-1-0716-3003-7}, issn = {1940-6029}, pages = {33--44}, publisher = {Springer Nature}, title = {{Molecular Cloning Using In Vivo DNA Assembly}}, doi = {10.1007/978-1-0716-3004-4_3}, volume = {2633}, year = {2023}, } @article{12759, abstract = {Stereological methods for estimating the 3D particle size and density from 2D projections are essential to many research fields. These methods are, however, prone to errors arising from undetected particle profiles due to sectioning and limited resolution, known as ‘lost caps’. A potential solution developed by Keiding, Jensen, and Ranek in 1972, which we refer to as the Keiding model, accounts for lost caps by quantifying the smallest detectable profile in terms of its limiting ‘cap angle’ (ϕ), a size-independent measure of a particle’s distance from the section surface. However, this simple solution has not been widely adopted nor tested. Rather, model-independent design-based stereological methods, which do not explicitly account for lost caps, have come to the fore. Here, we provide the first experimental validation of the Keiding model by comparing the size and density of particles estimated from 2D projections with direct measurement from 3D EM reconstructions of the same tissue. We applied the Keiding model to estimate the size and density of somata, nuclei and vesicles in the cerebellum of mice and rats, where high packing density can be problematic for design-based methods. Our analysis reveals a Gaussian distribution for ϕ rather than a single value. Nevertheless, curve fits of the Keiding model to the 2D diameter distribution accurately estimate the mean ϕ and 3D diameter distribution. While systematic testing using simulations revealed an upper limit to determining ϕ, our analysis shows that estimated ϕ can be used to determine the 3D particle density from the 2D density under a wide range of conditions, and this method is potentially more accurate than minimum-size-based lost-cap corrections and disector methods. Our results show the Keiding model provides an efficient means of accurately estimating the size and density of particles from 2D projections even under conditions of a high density.}, author = {Rothman, Jason Seth and Borges Merjane, Carolina and Holderith, Noemi and Jonas, Peter M and Angus Silver, R.}, issn = {1932-6203}, journal = {PLoS ONE}, number = {3 March}, publisher = {Public Library of Science}, title = {{Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy}}, doi = {10.1371/journal.pone.0277148}, volume = {18}, year = {2023}, } @article{12786, abstract = {AMPA glutamate receptors (AMPARs) mediate excitatory neurotransmission throughout the brain. Their signalling is uniquely diversified by brain region-specific auxiliary subunits, providing an opportunity for the development of selective therapeutics. AMPARs associated with TARP γ8 are enriched in the hippocampus, and are targets of emerging anti-epileptic drugs. To understand their therapeutic activity, we determined cryo-EM structures of the GluA1/2-γ8 receptor associated with three potent, chemically diverse ligands. We find that despite sharing a lipid-exposed and water-accessible binding pocket, drug action is differentially affected by binding-site mutants. Together with patch-clamp recordings and MD simulations we also demonstrate that ligand-triggered reorganisation of the AMPAR-TARP interface contributes to modulation. Unexpectedly, one ligand (JNJ-61432059) acts bifunctionally, negatively affecting GluA1 but exerting positive modulatory action on GluA2-containing AMPARs, in a TARP stoichiometry-dependent manner. These results further illuminate the action of TARPs, demonstrate the sensitive balance between positive and negative modulatory action, and provide a mechanistic platform for development of both positive and negative selective AMPAR modulators.}, author = {Zhang, Danyang and Lape, Remigijus and Shaikh, Saher A. and Kohegyi, Bianka K. and Watson, Jake and Cais, Ondrej and Nakagawa, Terunaga and Greger, Ingo H.}, issn = {2041-1723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics}}, doi = {10.1038/s41467-023-37259-5}, volume = {14}, 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}, } @article{13267, abstract = {Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure–function relationships of the brain’s complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.}, author = {Velicky, Philipp and Miguel Villalba, Eder and Michalska, Julia M and Lyudchik, Julia and Wei, Donglai and Lin, Zudi and Watson, Jake and Troidl, Jakob and Beyer, Johanna and Ben Simon, Yoav and Sommer, Christoph M and Jahr, Wiebke and Cenameri, Alban and Broichhagen, Johannes and Grant, Seth G.N. and Jonas, Peter M and Novarino, Gaia and Pfister, Hanspeter and Bickel, Bernd and Danzl, Johann G}, issn = {1548-7105}, journal = {Nature Methods}, pages = {1256--1265}, publisher = {Springer Nature}, title = {{Dense 4D nanoscale reconstruction of living brain tissue}}, doi = {10.1038/s41592-023-01936-6}, volume = {20}, year = {2023}, } @article{11951, abstract = {The mammalian hippocampal formation (HF) plays a key role in several higher brain functions, such as spatial coding, learning and memory. Its simple circuit architecture is often viewed as a trisynaptic loop, processing input originating from the superficial layers of the entorhinal cortex (EC) and sending it back to its deeper layers. Here, we show that excitatory neurons in layer 6b of the mouse EC project to all sub-regions comprising the HF and receive input from the CA1, thalamus and claustrum. Furthermore, their output is characterized by unique slow-decaying excitatory postsynaptic currents capable of driving plateau-like potentials in their postsynaptic targets. Optogenetic inhibition of the EC-6b pathway affects spatial coding in CA1 pyramidal neurons, while cell ablation impairs not only acquisition of new spatial memories, but also degradation of previously acquired ones. Our results provide evidence of a functional role for cortical layer 6b neurons in the adult brain.}, author = {Ben Simon, Yoav and Käfer, Karola and Velicky, Philipp and Csicsvari, Jozsef L and Danzl, Johann G and Jonas, Peter M}, issn = {2041-1723}, journal = {Nature Communications}, keywords = {General Physics and Astronomy, General Biochemistry, Genetics and Molecular Biology, General Chemistry, Multidisciplinary}, publisher = {Springer Nature}, title = {{A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory}}, doi = {10.1038/s41467-022-32559-8}, volume = {13}, year = {2022}, }