The molecular mechanisms underlying cellular processes occur at the nanoscale. The advent of super-resolution microscopy (SRI) in the past ten years (2014 Nobel Prize in chemistry) has enabled biologists to study these mechanisms with nanoscale precision in living systems, bridging the gap between electron microscopy and classical fluorescent microscopy, which resolution is inherently limited by the diffraction of light to ~250 nm. These methods have led to the discovery of major biological structures and cellular organizations, and new concepts are emerging from these studies, revealing discreet organizational patterns of biomolecules that are essential for their functions. In the particular context of brain transmission where neuronal contacts are established in a highly confined, dense, and restricted area of ~20 nm*250-500 nm, SRI opens new avenues to study these nano-environments, to ultimately understand how they are established and modulated throughout life. However, like all developing techniques in their early days, SRI has revealed important limitations. Whereas optical setups have been increasingly ameliorated to optimize imaging resolution, down to a few nanometers only (~ 10 nm), biologists still rely on traditional imaging tools such as large divalent antibodies, which sizes of ~15 nm * 2 (primary/secondary), defeat the purpose of increasing optical resolution down to 10 nm. Thus, the development of proper imaging tools that enable full benefit from these nanoscopic resolutions is still lacking, and efforts towards the generation of novel molecular tools to probe protein organization at the nanoscale is critical. In this project, we will: - Develop versatile 3-nm monomeric ligands, called “nanoprobes”, that will enable full benefit from the available optical resolutions, in order to gain novel information on the dynamics and nano-architecture of biomolecules in living cells and tissues - Fully functionalize and characterize these nanoprobes for 3D and quantitative super-resolution imaging and tissue labeling - Use the newly generated nanoprobes to decipher the dynamic nanoscale organization of synaptic proteins underlying the formation of brain connectivity. For probe development, libraries of genetically diversified small recombinant protein domains (~10 kDa) will be screened against specific epitopes and endogenous adhesion receptors involved in severe neurodevelopmental disease (Autistic spectrum disorders, schizophrenia, epilepsy). This will enable the generation of highly versatile nanoprobes as well as probes against endogenous cell receptors involved in synapse establishment and remodeling, to understand brain connectivity. The newly generated nanoprobes will be conjugated to state of the art fluorophores for in-depth 3D super-resolution imaging and to DNA oligonucleotides for use in a novel multi-protein imaging scheme, allowing to visualize multiple proteins within the same structure, with nanoscale precision (DNA-PAINT) and extract quantitative information (qPAINT). Using these efficient nanoprobes to target adhesion receptors and the diverse components of synaptic adhesions within neuronal cells and brain tissue, we will be able to decipher for the first time the internal organization of brain connections with maximal accuracy, providing 3D cartographies of their ultrastructures, and understand the impact of specific genetic mutations on this organization. Thus, this project is expected to lift two major barriers in the fields of i) fundamental neuroscience, by allowing to study the molecular nano-architecture and remodeling of brain connectivity in neuronal cultures and inside living tissues, an objective that has long been hampered by the lack of suitable tools for protein targeting in confined environments and ii) bio-imaging, by enabling scientists to benefit from optimized molecular tools that will allow versatile protein labeling and full accessible nanoscale resolution.

Synapses are complex protein-crowded micron-scale compartments allowing neuronal communication, whose dynamic changes in structure and molecular composition underlie essential brain functions. Among its key players, the adhesion protein neuroligin-1 (NLG1) is of special importance as it is critically placed at the synapse to interact with pre- and post-synaptic partners. Modulating NLG1 is known to perturb specific protein-protein interactions, resulting for instance in disorganizing the pre- and post-synaptic alignment. Mapping the nanoscale distribution of the proteins building the synapse in relation with dynamic morphological changes, especially when physiological modifications are induced, is important to truly understand how synapses work. We recently developed an innovative correlative nanoscopy platform allowing both Single-Molecule Localization Microscopy (SMLM) and extracellular space super-resolution microscopy (SUSHI-STED) to be performed on the same living biological sample. SMLM allows monitoring proteins organization and dynamics in living cells with down to a few nanometers spatial resolution while SUSHI-STED is a volumetric imaging technique particularly suited for imaging nanoscale cellular morphology. This correlative approach allows for mapping the nanoscale protein organization and dynamics within the densely arranged cellular structures, such as the connected pre- & post-synaptic compartments. Yet, several challenges still remain: i) multi-color single molecule localization and tracking is currently limited, ii) SUSHI-STED images cannot be easily segmented and iii) correlating and datamining the SMLM and SUSHI-STED quantifications necessitate heavy manual interaction. NANO-SYNATLAS aims at addressing these important issues by combining i) our recent correlative nanoscopy platform with ii) multicolor synaptic protein labelling for super-resolution microscopy, iii) genetic, optical, and pharmacological manipulation of synaptic components, and iv) advanced image segmentation and quantitative analysis. Good access to the dense synaptic cleft will be ensured by labeling proteins with a monomeric version of streptavidin that displays a small size (3 nm) and is compatible with 2-colors super-resolution microscopy. Three strategies will be used to perturb NLG1: i) genetic manipulation and ii) optogenetic control of NGL1; and iii) chemical protocols inducing long term potentiation or long term depression. Our correlative super-resolution microscopy platform will be operated to collect data of the (co)organization and dynamics of key synaptic components (adhesion, scaffold, cytoskeleton) with synapse morphology, for WT and perturbed NLG1. Deep-learning, geometry processing, and parametrization will be applied to automatically segment and classify synapses from the SUSHI-STED images. In parallel, cluster analysis and single molecule dynamics will be computed from the SMLM data. For each condition, an analytical model of the synapse will be created by projecting all the segmented SUSHI-STED synapses and SMLM quantifications. Each model will depict an atlas categorizing the diverse synapse states both in terms of morphology and internal structure. Their comparison will provide a better understanding on how specific protein-protein interactions regulate synapse dynamics and organization, with important insights on the underlying molecular mechanisms of neurodevelopmental pathologies such as autism spectrum disorders. The consortium is composed of the interdisciplinary teams “Quantitative Imaging of the Cell” (F. Levet/JB. Sibarita) and “Cell Adhesion Molecules In Synapse Assembly” (O. Thoumine), which have workd-wide recognized expertise in the fields of super-resolution microscopy, quantitative analysis, synapse biology, and fluorescent labelling approaches. This proposal captures a unique strategy to integrate our combined expertise to develop a next generation of quantitative tools for synapse biology.

Storage and recall of information depend on multiple forms of activity-dependent synaptic and intrinsic plasticity processes with distinct temporal dynamics. Within the hippocampus, a myriad of studies have focused on long-term changes in synaptic efficacy occurring on the postsynaptic side. In contrast, the cell-biological mechanisms and the functional consequences of presynaptic plasticity have been much less explored than postsynaptic forms of plasticity, in particular in relation to memory. We hypothesize that structural and functional plasticity on the presynaptic side, acting in a concerted fashion, shape the dynamics of neural processing in hippocampal circuits, which ultimately underlies mechanisms of memory encoding and recall. This line of thinking fills an important but largely unexplored research area, as previous studies considered either structural or functional plasticity in isolation but not both together, and have mostly focused on long-term postsynaptic forms of plasticity. Presynaptic forms of plasticity provide a highly dynamic mechanism to act on information transfer and circuit function, at different times scales ranging from hundreds of milliseconds to days. In addition, the morphology and stability of excitatory and inhibitory synapses (including presynaptic terminals) is highly variable over time, in particular in relation to learning. Here we will examine the mechanisms by which two essential presynaptic molecular components, Syt7 and GluK2, contribute to presynaptic plasticity at identified synapses in the hippocampus. In addition, we will investigate how the close interplay between presynaptic functional and structural plasticity determines the dynamics of information processing, encoding and retrieval in a memory-related brain region. We choose to focus on the CA3 region of the hippocampus, a key region for the early stages of memory acquisition, and in particular on DG-CA3 connections which constitute a major entry point from the cortex to the hippocampus. The following questions will be addressed: Aim 1. What are the subcellular mechanisms of short-term facilitation at DG-CA3 connections? Mf–CA3 PC synapses display a wide spectrum of functional plasticity and these connections serve as an excellent model for structure-function analyses of presynaptic plasticity. Aim 2. What are the dynamic features and mechanisms of structural plasticity at Mf-CA3 synapses? We want to clarify the activity-dependence of presynaptic structural plasticity, and understand the potential impact of memory encoding and processing on the morphology of Mf terminals. Aim 3. How do the two modalities of presynaptic plasticity combine to determine information processing and memory function in CA3? How does presynaptic plasticity impact information transfer and local CA3 circuit function (including excitation/inhibition balance) in vivo? What consequences on brain oscillations and brain state at a single cell and neural ensemble level? Is presynaptic plasticity involved in memory encoding and retrieval? The PREPLASH project is a basic science project aiming at understanding the mechanisms and functional consequences of presynaptic plasticity in a hippocampal circuit within the frame of memory. It is closely related to the themes of CES 16. Understanding the cellular and molecular mechanisms of neural plasticity remains one of the most important challenges for neuroscience research. Through the combined application of innovative experimental and analytical approaches, the project is likely to reveal new plasticity mechanisms, thereby fostering new insights into basic brain mechanisms. We are confident that the results obtained will be of high interest in the field of neuroscience, and will further fuel theories on brain mechanisms of information storage and recall

Decision-making often occurs in the absence of instruction to guide action. Instead, theories and experiments have predicted that the brain must compute a decision-value based on past experience to select the best action. This implies that the action with the highest subjective value should always be chosen. However, behavior is often stochastic with variability from trial-to-trial. To resolve this long-standing paradox, MOTORHEAD will take full advantage of state-of-the-art in vivo neuronal recordings and computational methods in rodent to bridge for the first time the gap between deterministic decision-signal and stochastic motor commands, achieving thus an unprecedented level of understanding of these “unpredictable” behaviors. Indeed, despite decade of intensive work, key questions remain unexplored: i) How such a deterministic decision signal is maintained without necessarily causing movement? ii) And how it is then converted to a biased motor command with trial-by-trial variability? Here, we hypothesize that these two operations occur across recurrent cortical layers of the secondary motor cortex (M2) of rodent, contributing to the proper balance between exploiting known secured options and exploring uncertain ones. Specifically, we posit that: i) Distinct populations of layer (L) 5 pyramidal neurons (PN) generate biased action according to the decision statistics provided by L2/3 PN. Specific attractor architectures, with different stability to noise, could cause the system to behave more or less randomly. ii) This top-down excitation could be gated by bottom-up plasticity forces from reward-related structures, which modulate decision-value to account for past choice outcome, notably when the action no longer generates the expected outcome. To achieve this breakthrough, we propose an ambitious system neuroscience approach, at high spatial/temporal resolution, to illuminate the cellular principles underlying the control and transformation of decision variable.

The dendritic spine, where the postsynaptic compartment of most excitatory synapses is localized, is a small protrusive structure emerging from the dendritic shaft. The molecular composition, the morphology and the compartmentalization of spines are critical for synaptic function. Moreover, spines are very plastic, they can appear and disappear in response to synaptic activity. Despite the importance of spine morphology and compartmentalization on synaptic function and plasticity, the molecular mechanisms according to which these structures are shaped and organized are not yet clear. The overarching objective of my project addresses these precise fundamental questions. I propose that β spectrins (2 and 3), two membrane bound actin filament cross-linking proteins, participate in setting spine morphology and compartmentalizing membrane proteins and that these functions involve their particular mechanical and lipid binding properties. The project is slip in three work packages and addresses these different aspects: (1) We will test the role of spectrins (2 and 3) on spine shape in baseline and during spine morphological changes. Morphological changes will be performed by physiological structural plasticity and innovative controlled spine deformation. A specific optical sensor will also allow us to assess directly spectrin tension in these different contexts. (2) We will investigate in detail β spectrins 2 and 3 nano and co-organization in dendritic spines. We will test the importance of their molecular partners including activity regulated lipids called phospholipids in their organizations and potential re-organization upon synaptic plasticity. (3) Finally, the dynamic compartmentalization of glutamate receptors is fundamental for synaptic function and several lines of evidence suggest that β spectrins could regulate their localization/stability in the spines. We will thus study the impact of β spectrins manipulation on synaptic functions, nano-organization and membrane dynamic of a major glutamate receptor, AMPA receptor. This grant will take full advantage of my PhD and my postdoctoral experiences in the exciting context of my new host laboratory, combining: innovative molecular tools, biophysics, electrophysiology and state-of-the-art imaging methods.