Synaptic responses adapt on millisecond-to-second timescales through short-term plasticity (STP), a key process that filters and transforms neuronal information. While STP is classically ascribed to presynaptic release mechanisms, postsynaptic receptor properties-particularly desensitization and surface diffusion-also shape synaptic responses. Here, we dissect pre- and postsynaptic contributions to synaptic adaptation using molecular tools to visualize glutamate release and manipulate AMPA receptor (AMPAR) diffusion in intact circuits. We find that synaptic gain during STP is tuned by synapse-specific regulation of AMPAR biophysics and diffusion-trapping. These features are determined constitutively by auxiliary subunit profiles and dynamically by activity-dependent signaling engaged during long-term plasticity. With modeling, we quantified how short-term synaptic dynamics are impacted by postsynaptic regulation of filtering properties, which broadened heterogeneity of filtering timescales to refine temporal selectivity in synaptic networks. By augmenting desensitization-mediated synaptic depression, AMPAR diffusion-trapping emerges as a fundamental regulatory mechanism of postsynaptic integration and circuit-level information processing.

Manganese is an essential trace metal for humans, but excessive exposure can cause neurotoxicity, including parkinsonian syndromes, cognitive deficits, and has also been implicated in the pathogenesis of neurodegenerative diseases. Globally, tens of millions of people are exposed to elevated manganese levels through drinking water, exceeding the WHO recommended guideline. Despite its public health significance, the cellular and subcellular mechanisms underlying manganese neurotoxicity remain poorly defined, particularly its distribution across brain cell types and its specific intracellular targets. In this study, we investigated manganese accumulation in primary cultures of hippocampal neurons and astrocytes. Using a correlative imaging approach that combined cryo-fluorescence light microscopy with synchrotron X-ray fluorescence imaging, we mapped and quantified manganese at subcellular resolution. Our analysis revealed that manganese preferentially accumulates in the Golgi apparatus of both neurons and astrocytes. In neurons, manganese was also detected in dendrites at the postsynaptic density, suggesting a role in synaptic vulnerability. Quantitative elemental analysis showed that astrocytes accumulated 3 times more manganese than neurons. Furthermore, when neurons were co-cultured with astrocytes, their manganese uptake was significantly reduced, indicating a possible protective or buffering role of astrocytes. These findings identify key cellular and subcellular targets of manganese and highlight the Golgi apparatus as a major regulator of manganese neurotoxicity. This work provides a foundation for understanding cell type–specific responses to manganese exposure and may inform the development of targeted neuroprotection strategies. Manganese neurotoxicity impact millions of people worldwide. Understanding how manganese distributes within brain cells is critical for addressing its neurotoxic effects. This study identifies the Golgi apparatus and postsynaptic density as key sites of manganese accumulation and reveals astrocytes as major regulators of neuronal manganese uptake. These findings provide a cellular framework for developing targeted strategies to mitigate manganese-induced neurotoxicity.

To meet the constantly improving spatial resolution offered by advanced microscopy techniques to study sub-cellular structures in biology, there is a need for small, monovalent probes that label proteins of interest with high specificity and minimal distance to the target, and are compatible with various imaging modalities. In this direction, we designed a strategy to generate minimal-size probes composed of a controlled 1:1 conjugate between a small domain binder and a 1.4 nm-gold nanoparticle with direct access to fluorescent labelling for dual light-electron microscopy. Our approach was applied to the widely used single-domain antibody against GFP (GBP). The modified GBP-gold conjugate retained normal binding to purified GFP in vitro, specifically labelled COS-7 cells and neurons expressing GFP-tagged synaptic membrane proteins, and penetrated readily into tight cell-cell contacts including neuronal synapses. The optional fluorescence labelling with a second ALFA nanobody allowed dSTORM imaging, while the silver-enhanced nanogold particle detected in TEM was used to characterize the number and nanoscale organization of individual proteins in the synaptic cleft. We counted a small number of endogenous neurexins in the pre-synapse and a larger number of AMPA receptors in the post-synapse, often aligned in nanodomains. This GBP-gold probe thus emerges as a potent tool to label an ever-increasing repertoire of GFP-tagged proteins in numerous biological organisms and models. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-CE11-0006-01, ANR-21-CE11-0019-01, ANR-21-CE44-0013, ANR-23-CE11-0007, ANR-10-INBS-04-0

A scarcity of live, paralog-specific tools has limited analysis of PSD-MAGUKs at excitatory synapses. To address this gap, we engineered small, 10FN3-derived binders that selectively recognize PSD-93 and SAP102 -alongside an enhanced PSD-95 reagent- and converted them into regulated, gene-encoded intrabodies for endogenous imaging. Through sequence-guided selection and targeted optimization, we obtained high-specificity reagents that label their native targets in neurons with minimal perturbation and support multiplexed live-cell and advanced imaging modalities. This toolkit enables differential visualization of MAGUK paralogs at native levels and provides a practical route to dissect their distinct contributions to synapse organization and plasticity. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-21-CE44-0013, ANR-16-CE16-0026, ANR-10-INBS-04, ANR-10-LABX-43

Synaptic responses adapt to fast repetitive inputs during bursts of neuronal network activity over timescales of milliseconds to seconds, either transiently facilitating or depressing. This high-frequency stimulus-dependent short-term synaptic plasticity (HF-STP) relies on a number of molecular processes that collectively endow synapses with filtering properties for information processing, optimized for the transmission of certain input frequencies and patterns in distinct circuits [1][1]–[3][2]. Changes in HF-STP are traditionally thought to stem from changes in pre-synaptic transmitter release [1][1],[2][3], but post-synaptic modifications in receptor biophysical properties or surface diffusion also regulate HF-STP [4][4]–[11][5]. A major challenge in understanding synapse function is to decipher how pre- and post-synaptic mechanisms synergistically tune synaptic transmission efficacy during HF-STP, and to determine how neuronal activity modifies post-synaptic signal computation and integration to diversify neuronal circuit function. Here, taking advantage of new molecular tools to directly visualize glutamate release [12][6] and specifically manipulate the surface diffusion of endogenous AMPAR in intact circuits [13][7], we define the respective contributions of pre-synaptic glutamate release, AMPAR desensitization and surface mobility to frequency-dependent synaptic adaptation. We demonstrate that post-synaptic gain control and signal integration capacity in synaptic networks is influenced by synapse-specific differences in AMPAR desensitization and diffusion-trapping characteristics that are shaped by molecular signaling events recruited during LTP. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-2 [4]: #ref-4 [5]: #ref-11 [6]: #ref-12 [7]: #ref-13

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.

Synapses are organized into nanocolumns that control synaptic transmission efficacy through precise alignment of postsynaptic neurotransmitter receptors and presynaptic release sites. Recent evidence show that Leucine-Rich Repeat Transmembrane protein LRRTM2, highly enriched and confined at synapses, interacts with Neurexins through its C-terminal cap, but the role of this binding interface has not been explored in synapse formation and function. Here, we develop a conditional knock-out mouse model (cKO) to address the molecular mechanisms of LRRTM2 regulation, and its role in synapse organization and function. We show that LRRTM2 cKO specifically impairs excitatory synapse formation and function in mice. Surface expression, synaptic clustering, and membrane dynamics of LRRTM2 are tightly controlled by selective motifs in the C-terminal domain. Conversely, the N-terminal domain controls presynapse nano-organization and postsynapse AMPAR sub-positioning and stabilization through the recently identified Neurexin-binding interface. Thus, we identify LRRTM2 as a central organizer of pre- and post- excitatory synapse nanostructure through interaction with presynaptic Neurexins.

The formation of adhesive cell-cell contacts is based on the intrinsic binding properties between specific transmembrane ligand-receptor pairs. In neurons, synaptic adhesion molecules provide a physical linkage between pre- and post-synaptic compartments, but the strength and the dynamic of these complexes in their actual membrane environments remain essentially unknown. To access such information, we developed a versatile assay to measure the affinity and binding kinetics of synaptic ligand-receptor interactions, based on the immobilization of Fc-tagged ligands on micropatterned substrates combined with live imaging of fluorescently-tagged counter receptors in heterologous cells. We applied this strategy to study the heterophilic complex formed between neurexin-1β (Nrx1β) and neuroligin-1 (Nlg1), compared to the homophilic SynCAM1 complex. First, the control of ligand density combined to the measurement of steady-state receptor enrichment at micropatterns demonstrates the high specificity of the matching molecular interactions and allows for the quantification of the two-dimensional affinity of the interaction in a membrane environment. Second, long-term FRAP experiments performed on the two molecular complexes and fitted with analytical models, demonstrate a diffusion-limited regime for SynCAM1 and a reaction-limited regime for Nlg1. This analysis provides a very long bond lifetime of the Nrx1β-Nlg1 complex, which by comparison with a monomeric mutant of Nlg1, can be attributed to the constitutive dimerization of Nlg1. Finally, we used the stable Nrx1β-Nlg1 complex as a pseudo-synaptic platform to analyze the rapid binding kinetics between the scaffolding protein PSD-95 and the intracellular domain of Nlg1, dissecting the contribution of the different PDZ domains through the use of specific PSD-95 point mutants.### Competing Interest StatementThe authors have declared no competing interest.

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.

Neuroligins (NLGNs) form a family of cell adhesion molecules implicated in synapse development, but the mechanisms that retain these proteins at synapses are still incompletely understood. Recent studies indicate that surface-associated NLGN1 is diffusionally trapped at synapses, where it interacts with quasi-static scaffolding elements of the post-synaptic density. Whereas single molecule tracking reveals rapid diffusion and transient immobilization of NLGN1 at synapses within seconds, fluorescence recovery after photobleaching experiments indicate instead a long-term turnover of NLGN1 at synapse, in the hour time range. To gain insight into the mechanisms supporting NLGN1 anchorage at post-synapses and try to reconcile those experimental paradigms, we quantitatively analyzed here live-cell and super-resolution imaging experiments performed on NLGN1 using a newly released simulator of membrane protein dynamics for fluorescence microscopy, FluoSim. Based on a small set of parameters including diffusion coefficients, binding constants, and photophysical rates, the framework describes fairly well the dynamic behavior of extra-synaptic and synaptic NLGN1 over both short and long time ranges, and provides an estimate of NLGN1 copy numbers in post-synaptic densities at steady-state (around 50 dimers). One striking result is that the residence time of NLGN1 at synapses is much longer than what can be expected from extracellular interactions with pre-synaptic neurexins only, suggesting that NLGN1 is stabilized at synapses through multivalent interactions with intracellular post-synaptic scaffolding proteins.

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.

Leucine Rich Repeat Transmembrane proteins (LRRTMs) are neuronal cell adhesion molecules involved in synapse development and plasticity. LRRTM2 is the most synaptogenic isoform of the family, and its expression is strongly restricted to excitatory synapses in mature neurons. However, the mechanisms by which LRRTM2 is trafficked and stabilized at synapses remain unknown. Here, we examine the role of LRRTM2 intracellular domain on its membrane expression and stabilization at excitatory synapses, using a knock-down strategy combined to single molecule tracking and super-resolution dSTORM microscopy. We show that LRRTM2 operates an important shift in mobility after synaptogenesis in hippocampal neurons. Knock-down of LRRTM2 during synapse formation reduced excitatory synapse density in mature neurons. Deletion of LRRTM2 C-terminal domain abolished the compartmentalization of LRRTM2 in dendrites and disrupted its synaptic enrichment. Furtheremore, we show that LRRTM2 diffusion is increased in the absence of its intracellular domain, and that the protein is more dispersed at synapses. Surprisingly, LRRTM2 confinement at synapses was strongly dependent on a YxxC motif in the C-terminal domain, but was independent of the PDZ-like binding motif ECEV. Finally, the nanoscale organization of LRRTM2 at excitatory synapses depended on its C-terminal domain, with involvement of both the PDZ-binding and YxxC motifs. Altogether, these results demonstrate that LRRTM2 trafficking and enrichment at excitatory synapses are dependent on its intracellular domain.

The brain is arguably the most complex organ. The branched and extended morphology of nerve cells, their subcellular complexity, the multiplicity of brain cell types as well as their intricate connectivity and the scattering properties of brain tissue present formidable challenges to the understanding of brain function. Neuroscientists have often been at the forefront of technological and methodological developments to overcome these hurdles to visualize, quantify and modify cell and network properties. Over the last few decades, the development of advanced imaging methods has revolutionized our approach to explore the brain. Super-resolution microscopy and tissue imaging approaches have recently exploded. These instrumentation-based innovations have occurred in parallel with the development of new molecular approaches to label protein targets, to evolve new biosensors and to target them to appropriate cell types or subcellular compartments. We review the latest developments for labelling and functionalizing proteins with small localization and functionalized reporters. We present how these molecular tools are combined with the development of a wide variety of imaging methods that break either the diffraction barrier or the tissue penetration depth limits. We put these developments in perspective to emphasize how they will enable step changes in our understanding of the brain.

Actin filaments generate mechanical forces that drive membrane movements during trafficking, endocytosis and cell migration. Reciprocally, adaptations of actin networks to forces regulate their assembly and architecture. Yet, a demonstration of forces acting on actin regulators at actin assembly sites in cells is missing. Here we show that local forces arising from actin filament elongation mechanically control WAVE regulatory complex (WRC) dynamics and function, that is, Arp2/3 complex activation in the lamellipodium. Single-protein tracking revealed WRC lateral movements along the lamellipodium tip, driven by elongation of actin filaments and correlating with WRC turnover. The use of optical tweezers to mechanically manipulate functional WRC showed that piconewton forces, as generated by single-filament elongation, dissociated WRC from the lamellipodium tip. WRC activation correlated with its trapping, dwell time and the binding strength at the lamellipodium tip. WRC crosslinking, hindering its mechanical dissociation, increased WRC dwell time and Arp2/3-dependent membrane protrusion. Thus, forces generated by individual actin filaments on their regulators can mechanically tune their turnover and hence activity during cell migration.

ABSTRACT 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 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.

Adhesion proteins play crucial roles at synapses, not only by providing a physical trans-synaptic linkage between axonal and dendritic membranes, but also by connecting to functional elements including the pre-synaptic neurotransmitter release machinery and post-synaptic receptors. To mediate these functions, adhesion proteins must be organized on the neuronal surface in a precise and controlled manner. Recent studies have started to describe the mobility, nanoscale organization, and turnover rate of key synaptic adhesion molecules including cadherins, neurexins, neuroligins, SynCAMs, and LRRTMs, and show that some of these proteins are highly mobile in the plasma membrane while others are confined at sub-synaptic compartments, providing evidence for different regulatory pathways. In this review article, we provide a biophysical view of the diffusional trapping of adhesion molecules at synapses, involving both extracellular and intracellular protein interactions. We review the methodology underlying these measurements, including biomimetic systems with purified adhesion proteins, means to perturb protein expression or function, single molecule imaging in cultured neurons, and analytical models to interpret the data. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts. We first achieve specificity toward a region outside of the interface by using phage display selection coupled with molecular and cellular validation. Highly selective competition is then generated by appending the more degenerate interaction peptide to contact the target interface. We apply this approach to specifically bind a single PDZ domain within the postsynaptic protein PSD-95 over highly similar PDZ domains in PSD-93, SAP-97 and SAP-102. Our work provides a paralog-selective and domain specific inhibitor of PSD-95, and describes a method to efficiently target other conserved PPI modules.