@article{22229,
abstract = {Hippocampal CA3 pyramidal neurons (PNs) form the largest autoassociative network in the mammalian brain. Whether CA3–CA3 recurrent connectivity is genetically preconfigured or environmentally shaped during ongoing memory storage is currently unknown. To address this question, we performed multicellular patch-clamp-based circuit mapping of up to eight CA3 PNs in the mouse hippocampus at multiple postnatal time points (P7–8, P18–25, and P45–50). Here, we show that the hippocampal CA3 network undergoes a developmental transformation from local, dense, and random connectivity to a distributed, sparse, and structured configuration. Thus, sparse and structured connectivity may emerge via experience-dependent mechanisms. In parallel, the strength of single synapses is downregulated; single synaptic events are sufficient to trigger postsynaptic spiking early in development, whereas spatial summation of several inputs is required at later time points. Biologically inspired models of memory storage by Hebbian synaptic plasticity and retrieval via pattern completion suggest that developmental changes improve specific aspects of memory storage and retrieval. Our results imply a developmental transformation of the neuronal code and the memory functions in the hippocampal CA3 network.},
author = {Vargas Barroso, Victor M and Watson, Jake and Navas Olivé, Andrea C and Schlögl, Alois and Jonas, Peter M},
issn = {2041-1723},
journal = {Nature Communications},
publisher = {Springer Nature},
title = {{Developmental emergence of sparse and structured synaptic connectivity in the hippocampal CA3 memory circuit}},
doi = {10.1038/s41467-026-71914-x},
volume = {17},
year = {2026},
}
@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},
}
@article{20099,
abstract = {The hippocampus, critical for learning and memory, is dogmatically described as a trisynaptic circuit where dentate gyrus granule cells (GCs), CA3 pyramidal neurons (PNs), and CA1 PNs are serially connected. However, CA3 also forms an autoassociative network, and its PNs have diverse morphologies, intrinsic properties, and GC input levels. How PN subtypes compose this recurrent network is unknown. To determine the synaptic arrangement of identified CA3 PNs, we combine multicellular patch-clamp recording and post hoc morphological analysis in mouse hippocampal slices. PNs can be divided into distinct “superficial” and “deep” subclasses, the latter including previously reported “athorny” cells. Subclasses have distinct input-output transformations and asymmetric connectivity, which is more abundant from superficial to deep PNs, splitting CA3 locally into two parallel recurrent networks. Coincident spontaneous inhibition occurs frequently within but not between subclasses, implying subclass-specific inhibitory innervation. Our results suggest two separately controlled sublayers for parallel information processing in hippocampal CA3.},
author = {Watson, Jake and Vargas Barroso, Victor M and Jonas, Peter M},
issn = {2211-1247},
journal = {Cell Reports},
number = {8},
publisher = {Elsevier},
title = {{Cell-specific wiring routes information flow through hippocampal CA3}},
doi = {10.1016/j.celrep.2025.116080},
volume = {44},
year = {2025},
}
@article{20457,
abstract = {Patch-clamp recording of miniature postsynaptic currents (mPSCs, or ‘minis’) is used extensively to investigate the functional properties of synapses. With this approach, spontaneous synaptic transmission events are recorded in an attempt to determine quantal synaptic parameters or the effect of synaptic manipulations. However, at the majority of brain synapses these events are small, with many undetectable due to recording noise. The effects of incomplete detection were well appreciated in the early years of synaptic physiology analysis, but appear to be increasingly forgotten. Here we sought to characterise the consequences of incomplete detection on the interpretability of mini analysis, using simulated mPSC data to give full control over event parameters. We demonstrate that commonly reported measures such as mean event amplitude and frequency, are misrepresented by the loss of undetected events. Probabilistic loss of small events results in detected event amplitude distributions that appear biologically complete, yet do not reflect the underlying synaptic properties. With both simulated and experimental datasets, we demonstrate that specific changes in event amplitude are primarily detected as changes in frequency, compromising classical biological interpretations. To facilitate more robust data analysis and interpretation, we detail a means for experimental estimation of the event detection limit and provide practical recommendations for data analysis. Together, our study highlights how mini analysis is prone to falsely reporting synaptic changes, raising awareness of these considerations, and provides a framework for more robust data analysis and interpretation.},
author = {Greger, Ingo H. and Watson, Jake},
issn = {1469-7793},
journal = {Journal of Physiology},
number = {22},
pages = {7189--7205},
publisher = {Wiley},
title = {{‘Mini analysis’ misrepresents changes in synaptic properties due to incomplete event detection}},
doi = {10.1113/JP288183},
volume = {603},
year = {2025},
}
@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{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},
}
@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},
}
@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{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},
}
@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{10763,
abstract = {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.},
author = {Herguedas, Beatriz and Kohegyi, Bianka K. and Dohrke, Jan Niklas and Watson, Jake and Zhang, Danyang and Ho, Hinze and Shaikh, Saher A. and Lape, Remigijus and Krieger, James M. and Greger, Ingo H.},
issn = {2041-1723},
journal = {Nature Communications},
publisher = {Springer Nature},
title = {{Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor}},
doi = {10.1038/s41467-022-28404-7},
volume = {13},
year = {2022},
}
@unpublished{11943,
abstract = {Complex wiring between neurons underlies the information-processing network enabling all brain functions, including cognition and memory. For understanding how the network is structured, processes information, and changes over time, comprehensive visualization of the architecture of living brain tissue with its cellular and molecular components would open up major opportunities. However, electron microscopy (EM) provides nanometre-scale resolution required for full in-silico reconstruction1–5, yet is limited to fixed specimens and static representations. Light microscopy allows live observation, with super-resolution approaches6–12 facilitating nanoscale visualization, but comprehensive 3D-reconstruction of living brain tissue has been hindered by tissue photo-burden, photobleaching, insufficient 3D-resolution, and inadequate signal-to-noise ratio (SNR). Here we demonstrate saturated reconstruction of living brain tissue. We developed an integrated imaging and analysis technology, adapting stimulated emission depletion (STED) microscopy6,13 in extracellularly labelled tissue14 for high SNR and near-isotropic resolution. Centrally, a two-stage deep-learning approach leveraged previously obtained information on sample structure to drastically reduce photo-burden and enable automated volumetric reconstruction down to single synapse level. Live reconstruction provides unbiased analysis of tissue architecture across time in relation to functional activity and targeted activation, and contextual understanding of molecular labelling. This adoptable technology will facilitate novel insights into the dynamic functional architecture of living brain tissue.},
author = {Velicky, Philipp and Miguel Villalba, Eder and Michalska, Julia M 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},
booktitle = {bioRxiv},
title = {{Saturated reconstruction of living brain tissue}},
doi = {10.1101/2022.03.16.484431},
year = {2022},
}
@unpublished{11950,
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 nanoscopic synaptic scales in diverse chemically fixed brain preparations, including rodent and human. CATS leverages fixation-compatible extracellular labeling and advanced optical readout, in particular stimulated-emission depletion and expansion microscopy, to comprehensively delineate cellular structures. It enables 3D-reconstructing single synapses and mapping synaptic connectivity by identification and tailored analysis of putative synaptic cleft regions. Applying CATS to the hippocampal mossy fiber circuitry, we demonstrate its power to reveal the system’s molecularly informed ultrastructure across spatial scales and assess local connectivity by reconstructing and quantifying the synaptic input and output structure of identified neurons.},
author = {Michalska, Julia M and Lyudchik, Julia and Velicky, Philipp and Korinkova, Hana and Watson, Jake and Cenameri, Alban and Sommer, Christoph M and Venturino, Alessandro and Roessler, Karl and Czech, Thomas and Siegert, Sandra and Novarino, Gaia and Jonas, Peter M and Danzl, Johann G},
booktitle = {bioRxiv},
title = {{Uncovering brain tissue architecture across scales with super-resolution light microscopy}},
doi = {10.1101/2022.08.17.504272},
year = {2022},
}
@article{9549,
abstract = {AMPA receptors (AMPARs) mediate the majority of excitatory transmission in the brain and enable the synaptic plasticity that underlies learning1. A diverse array of AMPAR signalling complexes are established by receptor auxiliary subunits, which associate with the AMPAR in various combinations to modulate trafficking, gating and synaptic strength2. However, their mechanisms of action are poorly understood. Here we determine cryo-electron microscopy structures of the heteromeric GluA1–GluA2 receptor assembled with both TARP-γ8 and CNIH2, the predominant AMPAR complex in the forebrain, in both resting and active states. Two TARP-γ8 and two CNIH2 subunits insert at distinct sites beneath the ligand-binding domains of the receptor, with site-specific lipids shaping each interaction and affecting the gating regulation of the AMPARs. Activation of the receptor leads to asymmetry between GluA1 and GluA2 along the ion conduction path and an outward expansion of the channel triggers counter-rotations of both auxiliary subunit pairs, promoting the active-state conformation. In addition, both TARP-γ8 and CNIH2 pivot towards the pore exit upon activation, extending their reach for cytoplasmic receptor elements. CNIH2 achieves this through its uniquely extended M2 helix, which has transformed this endoplasmic reticulum-export factor into a powerful AMPAR modulator that is capable of providing hippocampal pyramidal neurons with their integrative synaptic properties. },
author = {Zhang, Danyang and Watson, Jake and Matthews, Peter M. and Cais, Ondrej and Greger, Ingo H.},
issn = {1476-4687},
journal = {Nature},
pages = {454--458},
publisher = {Springer Nature},
title = {{Gating and modulation of a hetero-octameric AMPA glutamate receptor}},
doi = {10.1038/s41586-021-03613-0},
volume = {594},
year = {2021},
}
@article{9985,
abstract = {AMPA receptor (AMPAR) abundance and positioning at excitatory synapses regulates the strength of transmission. Changes in AMPAR localisation can enact synaptic plasticity, allowing long-term information storage, and is therefore tightly controlled. Multiple mechanisms regulating AMPAR synaptic anchoring have been described, but with limited coherence or comparison between reports, our understanding of this process is unclear. Here, combining synaptic recordings from mouse hippocampal slices and super-resolution imaging in dissociated cultures, we compare the contributions of three AMPAR interaction domains controlling transmission at hippocampal CA1 synapses. We show that the AMPAR C-termini play only a modulatory role, whereas the extracellular N-terminal domain (NTD) and PDZ interactions of the auxiliary subunit TARP γ8 are both crucial, and each is sufficient to maintain transmission. Our data support a model in which γ8 accumulates AMPARs at the postsynaptic density, where the NTD further tunes their positioning. This interplay between cytosolic (TARP γ8) and synaptic cleft (NTD) interactions provides versatility to regulate synaptic transmission and plasticity.},
author = {Watson, Jake and Pinggera, Alexandra and Ho, Hinze and Greger, Ingo H.},
issn = {2041-1723},
journal = {Nature Communications},
number = {1},
publisher = {Nature Publishing Group},
title = {{AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions}},
doi = {10.1038/s41467-021-25281-4},
volume = {12},
year = {2021},
}