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 AU - Chen, JingJing ID - 15101 SN - 2663 - 337X TI - Developmental transformation of nanodomain coupling between Ca2+ channels and release sensors at a central GABAergic synapse ER - TY - JOUR AB - 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. AU - Vandael, David H AU - Jonas, Peter M ID - 15117 IS - 6687 JF - Science TI - Structure, biophysics, and circuit function of a "giant" cortical presynaptic terminal VL - 383 ER - TY - CHAP AB - 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. AU - Arroyo-Urea, Sandra AU - Watson, Jake AU - García-Nafría, Javier ED - Scarlett, Garry ID - 12720 SN - 1064-3745 T2 - DNA Manipulation and Analysis TI - Molecular Cloning Using In Vivo DNA Assembly VL - 2633 ER - TY - JOUR AB - 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. AU - Mrestani, Achmed AU - Lichter, Katharina AU - Sirén, Anna Leena AU - Heckmann, Manfred AU - Paul, Mila M. AU - Pauli, Martin ID - 12567 IS - 3 JF - International Journal of Molecular Sciences TI - Single-molecule localization microscopy of presynaptic active zones in Drosophila melanogaster after rapid cryofixation VL - 24 ER - TY - JOUR AB - 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. AU - Rothman, Jason Seth AU - Borges Merjane, Carolina AU - Holderith, Noemi AU - Jonas, Peter M AU - Angus Silver, R. ID - 12759 IS - 3 March JF - PLoS ONE TI - Validation of a stereological method for estimating particle size and density from 2D projections with high accuracy VL - 18 ER - TY - JOUR AB - 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. AU - Ortiz-Leal, Irene AU - Torres, Mateo V. AU - Vargas Barroso, Victor M AU - Fidalgo, Luis Eusebio AU - López-Beceiro, Ana María AU - Larriva-Sahd, Jorge A. AU - Sánchez-Quinteiro, Pablo ID - 12515 JF - Frontiers in Neuroanatomy TI - The olfactory limbus of the red fox (Vulpes vulpes). New insights regarding a noncanonical olfactory bulb pathway VL - 16 ER - TY - JOUR AB - 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. AU - Zhang, Danyang AU - Lape, Remigijus AU - Shaikh, Saher A. AU - Kohegyi, Bianka K. AU - Watson, Jake AU - Cais, Ondrej AU - Nakagawa, Terunaga AU - Greger, Ingo H. ID - 12786 JF - Nature Communications TI - Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics VL - 14 ER - TY - JOUR AB - 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. AU - Velicky, Philipp AU - Miguel Villalba, Eder AU - Michalska, Julia M AU - Lyudchik, Julia AU - Wei, Donglai AU - Lin, Zudi AU - Watson, Jake AU - Troidl, Jakob AU - Beyer, Johanna AU - Ben Simon, Yoav AU - Sommer, Christoph M AU - Jahr, Wiebke AU - Cenameri, Alban AU - Broichhagen, Johannes AU - Grant, Seth G.N. AU - Jonas, Peter M AU - Novarino, Gaia AU - Pfister, Hanspeter AU - Bickel, Bernd AU - Danzl, Johann G ID - 13267 JF - Nature Methods SN - 1548-7091 TI - Dense 4D nanoscale reconstruction of living brain tissue VL - 20 ER -