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Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD. Long-term depression (LTD) of synaptic strength contributes to circuit remodeling, memory encoding and erasure. Here, the authors show that P2XR- and NMDAR-dependent LTD are associated with distinct and precise molecular modifications that lead to specific modification of synapse function.

The nanoscale organization of neurotransmitter receptors regarding pre-synaptic release sites is a fundamental determinant of the synaptic transmission amplitude and reliability. How modifications in the pre- and post-synaptic machinery alignments affects synaptic currents, has only been addressed with computer modelling. Using single molecule super-resolution microscopy, we found a strong spatial correlation between AMPA receptor (AMPAR) nanodomains and the post-synaptic adhesion protein neuroligin-1 (NLG1). Expression of a truncated form of NLG1 disrupted this correlation without affecting the intrinsic AMPAR organization, shifting the pre-synaptic release machinery away from AMPAR nanodomains. Electrophysiology in dissociated and organotypic hippocampal rodent cultures shows these treatments significantly decrease AMPAR-mediated miniature and EPSC amplitudes. Computer modelling predicts that ~100 nm lateral shift between AMPAR nanoclusters and glutamate release sites induces a significant reduction in AMPAR-mediated currents. Thus, our results suggest the synapses necessity to release glutamate precisely in front of AMPAR nanodomains, to maintain a high synaptic responses efficiency.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.

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.

Plasticity at excitatory synapses can be induced either by synaptic release of glutamate or the release of gliotransmitters such as ATP. Recently, we showed that postsynaptic P2X2 receptors activated by ATP released from astrocytes downregulate synaptic AMPAR, providing a novel mechanism by which glial cells modulate synaptic activity. ATP- and lNMDA-induced depression in the CA1 region of the hippocampus are additive, suggesting distinct molecular pathways. AMPARs are homo-or hetero-tetramers composed of GluA1-A4. Here, we first show that P2X2-mediated AMPAR inhibition is dependent on the subunit composition of AMPAR. GluA3 homomers are insensitive and their presence in heteromers alters P2X-mediated inhibition. Using a mutational approach, we demonstrate that the two CaMKII phosphorylation sites S567 and S831 located in the cytoplasmic Loop1 and C-terminal tail of GluA1 subunits, respectively, are critical for P2X2-mediated AMPAR inhibition recorded from co-expressing Xenopus oocytes and removal of surface AMPAR at synapses of hippocampal neurons imaged by the super-resolution dSTORM technique. Finally, using phosphorylation site-specific antibodies, we show that P2X-induced depression in hippocampal slices produces a dephosphorylation of the GluA1 subunit at S567, contrary to NMDAR-mediated LTD. These findings indicate that GluA1 phosphorylation of S567 and S831 is critical for P2X2-mediated AMPAR internalization and ATP-driven synaptic depression.

Chemical synapses control their strength through the nanoscale clustering of postsynaptic receptors into sub-synaptic domains (SSDs). Despite their importance in synapse function, the properties and plasticity of these domains are not well understood in vivo, particularly in inhibitory synapses. We used direct Stochastic Optical Resolution Microscopy (dSTORM) to show that Gephyrin, the main inhibitory receptor scaffold protein, is organised into SSDs in vivo, with distinct arrangements depending on their sub-cellular location and presynaptic partner. Furthermore, chronic chemogenetic increases in cortical activity caused a reduction in Gephyrin SSD volume specifically in axo-axonic, but not axo-dendritic, synapses. Functionally, this resulted in a weakening of axo-axonic contacts. We show that the nanoscale arrangement of synapses in the brain is plastic and used to fine-tune synaptic gain in vivo.Competing Interest StatementThe authors have declared no competing interest.Footnotes* Manuscript text and figures have been revised.

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from P21 to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared to neighbouring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favour synchronous activity in CA1 networks.

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.

Long-Term Potentiation (LTP) and Long-Term Depression (LTD) of excitatory synaptic transmission are considered as cellular basis of learning and memory. These two forms of synaptic plasticity have been mainly attributed to global changes in the number of synaptic AMPA-type glutamate receptor (AMPAR) through a regulation of the diffusion/trapping balance at the PSD, exocytosis and endocytosis. While the precise molecular mechanisms at the base of LTP have been intensively investigated, the ones involved in LTD remains elusive. Here we combined super-resolution imaging technique, electrophysiology and modeling to describe the various modifications of AMPAR nanoscale organization and their effect on synaptic transmission in response of two different LTD protocols, based on the activation of either NMDA receptors or P2X receptors. While both LTDs are associated with a decrease in synaptic AMPAR clustering, only NMDAR-dependent LTD is associated with a reorganization of PSD95 at the nanoscale. This change increases the pool of diffusive AMPAR improving synaptic short-term facilitation through a post-synaptic mechanism. These results demonstrate that specific dynamic reorganization of synapses at the nanoscale during specific LTD paradigm allows to improve the responsiveness of depressed synapses.