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 - JOUR AB - AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance. AU - Herguedas, Beatriz AU - Kohegyi, Bianka K. AU - Dohrke, Jan Niklas AU - Watson, Jake AU - Zhang, Danyang AU - Ho, Hinze AU - Shaikh, Saher A. AU - Lape, Remigijus AU - Krieger, James M. AU - Greger, Ingo H. ID - 10763 JF - Nature Communications TI - Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor VL - 13 ER - TY - JOUR AB - 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. AU - Ben Simon, Yoav AU - Käfer, Karola AU - Velicky, Philipp AU - Csicsvari, Jozsef L AU - Danzl, Johann G AU - Jonas, Peter M ID - 11951 JF - Nature Communications KW - General Physics and Astronomy KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Chemistry KW - Multidisciplinary SN - 2041-1723 TI - A direct excitatory projection from entorhinal layer 6b neurons to the hippocampus contributes to spatial coding and memory VL - 13 ER - TY - JOUR AB - To understand the function of neuronal circuits, it is crucial to disentangle the connectivity patterns within the network. However, most tools currently used to explore connectivity have low throughput, low selectivity, or limited accessibility. Here, we report the development of an improved packaging system for the production of the highly neurotropic RVdGenvA-CVS-N2c rabies viral vectors, yielding titers orders of magnitude higher with no background contamination, at a fraction of the production time, while preserving the efficiency of transsynaptic labeling. Along with the production pipeline, we developed suites of ‘starter’ AAV and bicistronic RVdG-CVS-N2c vectors, enabling retrograde labeling from a wide range of neuronal populations, tailored for diverse experimental requirements. We demonstrate the power and flexibility of the new system by uncovering hidden local and distal inhibitory connections in the mouse hippocampal formation and by imaging the functional properties of a cortical microcircuit across weeks. Our novel production pipeline provides a convenient approach to generate new rabies vectors, while our toolkit flexibly and efficiently expands the current capacity to label, manipulate and image the neuronal activity of interconnected neuronal circuits in vitro and in vivo. AU - Sumser, Anton L AU - Jösch, Maximilian A AU - Jonas, Peter M AU - Ben Simon, Yoav ID - 12288 JF - eLife KW - General Immunology and Microbiology KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Medicine KW - General Neuroscience TI - Fast, high-throughput production of improved rabies viral vectors for specific, efficient and versatile transsynaptic retrograde labeling VL - 11 ER -