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This review focuses on the activity-dependent diffusion trapping of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) as a crucial mechanism for the expression of early long-term potentiation (LTP), a process central to learning and memory. Despite decades of research, the precise mechanisms by which LTP induction leads to an increase in AMPAR responses at synapses have been elusive. We review the different hypotheses that have been put forward to explain the increased AMPAR responsiveness during LTP. We discuss the dynamic nature of AMPAR complexes, including their constant turnover and activity-dependent modifications that affect their synaptic accumulation. We highlight a hypothesis suggesting that AMPARs are diffusively trapped at synapses through activity-dependent interactions with protein-based binding slots in the post-synaptic density (PSD), offering a potential explanation for the increased synaptic strength during LTP. Furthermore, we outline the challenges still to be addressed before we fully understand the functional roles and molecular mechanisms of AMPAR dynamic nanoscale organization in LTP. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

The mode of action of a synaptic protein is challenged.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques., Competing Interests: CR, CB, BC, ET, FV, MF, PM, CG, VP, IG, EH, SC, GG, IC, CM, CP, DC, MS No competing interests declared, (© 2024, Rimbault, Breillat, Compans et al.)

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada., (© 2024 The Authors.)

Correlative light and electron microscopy (CLEM) can provide valuable information about a biological sample by giving information on the specific localization of a molecule of interest within an ultrastructural context. In this work, we describe a simple CLEM method to obtain high-resolution images of neurotransmitter receptor distribution in synapses by electron microscopy (EM). We use hippocampal organotypic slices from a previously reported mouse model expressing a modified AMPA receptor (AMPAR) subunit that binds biotin at the surface (Getz et al., 2022). This tag can be recognized by StreptAvidin-Fluoronanogold™ conjugates (SA-FNG), which reach receptors at synapses (synaptic cleft is 50-100nm thick). By using pre-embedding labeling, we found that SA-FNG reliably bind synaptic receptors and penetrate around 10-15μm in depth in live tissue. However, the silver enhancement was only reaching the surface of the slices. We show that permeabilization with triton is highly effective at increasing the in depth-gold amplification and that the membrane integrity is well preserved. Finally, we also apply high-resolution electron tomography, thus providing important information about the 3D organization of surface AMPA receptors in synapses at the nanoscale., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

The consolidation of recent memories depends on memory replays, also called ripples, generated within the hippocampus during slow-wave sleep, and whose inactivation leads to memory impairment. For now, the mobilisation, localisation and importance of synaptic plasticity events associated to ripples are largely unknown. To tackle this question, we used cell surface AMPAR immobilisation to block post-synaptic LTP within the hippocampal region of male mice during a spatial memory task, and show that: 1- hippocampal synaptic plasticity is engaged during consolidation, but is dispensable during encoding or retrieval. 2- Plasticity blockade during sleep results in apparent forgetting of the encoded rule. 3- In vivo ripple recordings show a strong effect of AMPAR immobilisation when a rule has been recently encoded. 4- In situ investigation suggests that plasticity at CA3-CA3 recurrent synapses supports ripple generation. We thus propose that post-synaptic AMPAR mobility at CA3 recurrent synapses is necessary for ripple-dependent rule consolidation., (© 2023. The Author(s).)

In the central nervous system, glutamatergic synapses play a central role in the regulation of excitatory neuronal transmission. With the membrane-associated guanylate kinase (MAGUK) family of proteins as their structuring scaffold, glutamatergic receptors serve as the powerhouse of glutamatergic synapses. Glutamatergic receptors can be categorized as metabotropic and ionotropic receptors. The latter are then categorized into N-methyl-d-aspartate, kainate receptors, and α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid receptors (AMPARs). Over the past two decades, genetic tagging technology and super-resolution microscopy have been of the utmost importance to unravel how the different receptors are organized at glutamatergic synapses. At the plasma membrane, receptors are highly mobile but show reduced mobility when at synaptic sites. This partial immobilization of receptors at synaptic sites is attributed to the stabilization/anchoring of receptors with the postsynaptic MAGUK proteins and auxiliary proteins, and presynaptic proteins. These partial immobilizations and localization of glutamatergic receptors within the synaptic sites are fundamental for proper basal transmission and synaptic plasticity. Perturbations of the stabilization of glutamatergic receptors are often associated with cognitive deficits. In this review, we describe the proposed mechanisms for synaptic localization and stabilization of AMPARs, the major players of fast excitatory transmission in the central nervous system., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Changes in the number of synaptic AMPA receptors underlie many forms of synaptic plasticity. These variations are controlled by an interplay between their intracellular transport (IT), export to the plasma membrane (PM), stabilization at synapses, and recycling. The cytosolic C-terminal domain of the AMPAR GluA1 subunit is specifically associated with 4.1 N and SAP97. We analyze how interactions between GluA1 and 4.1N or SAP97 regulate IT and exocytosis in basal conditions and after cLTP induction. The down-regulation of 4.1N or SAP97 decreases GluA1 IT properties and export to the PM. The total deletion of its C-terminal fully suppresses its IT. Our results demonstrate that during basal transmission, the binding of 4.1N to GluA1 allows their exocytosis whereas the interaction with SAP97 is essential for GluA1 IT. During cLTP, the interaction of 4.1N with GluA1 allows its IT and exocytosis. Our results identify the differential roles of 4.1N and SAP97 in the control of various phases of GluA1 IT., Competing Interests: CB, JC, NR, DC, FC No competing interests declared, (© 2023, Bonnet et al.)

Lattice light-sheet microscopy (LLSM) is a very efficient technique for high resolution 3D imaging of dynamic phenomena in living biological samples. However, LLSM imaging remains limited in depth due to optical aberrations caused by sample-based refractive index mismatch. Here, we propose a simple and low-cost active image optimization (AIO) method to recover high resolution imaging inside thick biological samples. AIO is based on (1) a light-sheet autofocus step (AF) followed by (2) an adaptive optics image-based optimization. We determine the optimum AIO parameters to provide a fast, precise and robust aberration correction on biological samples. Finally, we demonstrate the performances of our approach on sub-micrometric structures in brain slices and plant roots., Competing Interests: MM, FH, CV, JL: Imagine Optic (E), (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPARs) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminal. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP-GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

MDGA molecules can bind neuroligins and interfere with trans-synaptic interactions to neurexins, thereby impairing synapse development. However, the subcellular localization and dynamics of MDGAs, or their specific action mode in neurons remain unclear. Here, surface immunostaining of endogenous MDGAs and single molecule tracking of recombinant MDGAs in dissociated hippocampal neurons reveal that MDGAs are homogeneously distributed and exhibit fast membrane diffusion, with a small reduction in mobility across neuronal maturation. Knocking-down/out MDGAs using shRNAs and CRISPR/Cas9 strategies increases the density of excitatory synapses, the membrane confinement of neuroligin-1, and the phosphotyrosine level of neuroligins associated with excitatory post-synaptic differentiation. Finally, MDGA silencing reduces the mobility of AMPA receptors, increases the frequency of miniature EPSCs (but not IPSCs), and selectively enhances evoked AMPA-receptor-mediated EPSCs in CA1 pyramidal neurons. Overall, our results support a mechanism by which interactions between MDGAs and neuroligin-1 delays the assembly of functional excitatory synapses containing AMPA receptors., Competing Interests: AT, ML, GB, BT, SD, AF, IC, KV, JV, MS, Jd, DC, OT No competing interests declared

The accumulation of AMPARs to synapses is a fundamental step in Long-term potentiation (LTP) of synaptic transmission, a well-established cellular correlate of learning and memory. The discovery of a sizeable and highly mobile population of extrasynaptic AMPARs - randomly scanning the synaptic surface under basal conditions - provided a conceptual framework for a simplified model: LTP can be induced by the capture, and hence accumulation, of laterally diffusing extrasynaptic AMPARs. Here, we review the evidence supporting a rate-limiting role of AMPAR lateral diffusion in LTP and as consequence, in learning and memory. We propose that there are "multiple solutions" for achieving the diffusional trapping of AMPAR during LTP, mainly mediated by the interaction between interchangeable AMPAR auxiliary subunits and cell-adhesion molecules containing PDZ-binding domains and synaptic scaffolds containing PDZ-domains. We believe that this molecular degeneracy in the diffusional trapping of AMPAR during LTP serve to ensure the robustness of this crucial step in the making of memories. All in all, the role of AMPAR lateral diffusion in LTP is not only a conceptual leap in our understanding of memory, but it might also hold the keys for the development of therapeutics against disorders associated with memory deficits such as Alzheimer's disease., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The pruning of dendritic spines during development requires autophagy. This process is facilitated by long-term depression (LTD)-like mechanisms, which has led to speculation that LTD, a fundamental form of synaptic plasticity, also requires autophagy. Here, we show that the induction of LTD via activation of NMDA receptors or metabotropic glutamate receptors initiates autophagy in the postsynaptic dendrites in mice. Dendritic autophagic vesicles (AVs) act in parallel with the endocytic machinery to remove AMPA receptor subunits from the membrane for degradation. During NMDAR-LTD, key postsynaptic proteins are sequestered for autophagic degradation, as revealed by quantitative proteomic profiling of purified AVs. Pharmacological inhibition of AV biogenesis, or conditional ablation of atg5 in pyramidal neurons abolishes LTD and triggers sustained potentiation in the hippocampus. These deficits in synaptic plasticity are recapitulated by knockdown of atg5 specifically in postsynaptic pyramidal neurons in the CA1 area. Conducive to the role of synaptic plasticity in behavioral flexibility, mice with autophagy deficiency in excitatory neurons exhibit altered response in reversal learning. Therefore, local assembly of the autophagic machinery in dendrites ensures the degradation of postsynaptic components and facilitates LTD expression., (© 2022. The Author(s).)

During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children., (© The Author(s) 2021. Published by Oxford University Press.)

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution - typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy., (© 2021. The Author(s).)