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Learning and memory correlate with activity-dependent synaptic plasticity processes at appropriate synaptic circuits. The underlying mechanisms of information storage in the brain are currently investigated at a whole-neuron scale to identify cellular memory engrams i.e., ensembles of neurons whose recruitment and activation are necessary and sufficient for the retrieval of a specific memory. Traditional methods for structural and functional analysis of synapses are not sufficient for investigating which subset of synapses encodes and stores a specific memory in a given neuron. To address this fundamental question, we have developed ‘SynActive’, a genetic toolbox exploiting regulatory sequences from the Arc mRNA and synapse-targeting peptides, that allows the expression of any protein of interest specifically at potentiated synapses. Here I have extended the SynActive toolbox to express the protein of interest, including fluorescent reporters, an affinity purification tag, and an optogenetic actuator specifically at in vitro and in vivo potentiated spines. In SynActive-eGRASP, which allows input-specific labeling of potentiated synapses, one split-GFP fragment was expressed constitutively by presynaptic neurons, while the postsynaptic half was synthesized in an activity-dependent fashion at potentiated spines. After extensive validation in cultured neurons, SynActive-eGRASP was employed to map CA3-CA1 synapses potentiated during an associative memory task – contextual fear conditioning. Semi-automated analysis using a custom-made algorithm revealed a spatially nonuniform and clustered distribution of SynActive-eGRASP-positive synapses. SynActive controlled expression of fluorescent reporters- mVenus, and DsRED-E5 labeled dendritic spines undergoing potentiation in primary neuronal cultures. These optimized vectors should facilitate large-scale, possibly brain-wide as well as time-dependent, mapping of potentiated spines. For the proteomic profiling of in vivo potentiated spines, SynActive AAVs expressing FLAG-tagged PSD95 was delivered to the mouse hippocampus and the PSD95-interactome was immunoprecipitated from potentiated synapses after contextual fear conditioning. In primary neuronal cultures, photoactivation of channelrhodopsin expressed at potentiated spines via SynActive method induced neuronal spiking. In vivo, this construct can be used to tag memory-specific synapses, and optically activating them might induce memory retrieval. These novel tools and the initial results they produced provide the first step towards a shift in the study of memory engrams from a cellular to a synaptic resolution. In addition, our quantitative maps of synaptic potentiation in whole brain areas or specific synaptic circuits can be used to refine computational models of neural plasticity. Ongoing experiments are aimed at performing a comparative analysis of synaptic maps obtained in different phases of memory encoding and recall, in both physiological conditions and models of neurodegenerative and neurodevelopmental diseases.

of the oral communication presented at the Annual Retreat of the Institute of Neuroscience of the Italian National Research Council, held in Pisa, October 2-4, 2019.

of the poster presented at the joint meeting of SIF - Italian Physiological Society and FEPS - Federation of European Physiological Societies, held in Bologna, September 10-13, 2019.

of the poster presented at the "proMEMO - Proteins and Circuits in Memory" conference, held in Copenhagen, March 5-8, 2019.

from the oral communication at the Society for Neuroscience annual meeting held in San Diego (CA, USA), November 3-7, 2018. Authors M. Mainardi and F. Gobbo also co-chaired the nanosymposium "444 - LTP: Intracellular Signaling, Pre- and Post-synaptic mechanisms".

from the oral communication at the Society for Neuroscience annual meeting held in San Diego (CA, USA), November 3-7, 2018. Authors M. Mainardi and F. Gobbo also co-chaired the nanosymposium "444 - LTP: Intracellular Signaling, Pre- and Post-synaptic mechanisms".

Increasing evidence points to the importance of dendritic spines in the formation and allocation of memories, and alterations of spine number and physiology are associated to memory and cognitive disorders. Modifications of the activity of subsets of synapses are believed to be crucial for memory establishment. However, the development of a method to directly test this hypothesis, by selectively controlling the activity of potentiated spines, is currently lagging. Here we introduce a hybrid RNA/protein approach to regulate the expression of a light-sensitive membrane channel at activated synapses, enabling selective tagging of potentiated spines following the encoding of a novel context in the hippocampus. This approach can be used to map potentiated synapses in the brain and will make it possible to re-activate the neuron only at previously activated synapses, extending current neuron-tagging technologies in the investigation of memory processes., Changes to subsets of dendritic spines are thought to be important for memory formation. Here, the authors develop a hybrid RNA/protein tool that allows for optogenetic stimulation of single synapses that have been tagged in an activity-dependent manner

The p75 neurotrophin (NT) receptor (p75(NTR)) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75(NTR) activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75(NTR) in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75(NTR) are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75(NTR) and mutated (mut) p75(NTR) differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75(NTR), but not of mut p75(NTR), to mediate immature NT (proNT)-induced apoptosis. Both p75(NTR) forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multi-faceted activity of the p75(NTR) monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature.

Growth cone filopodia are actin-based mechanosensory structures essential for chemoreception and generation of contractile forces necessary for directional motility. However, little is known about the influence of filopodial actin structures on substrate adhesion and filopodial contractility. Formin-2 (Fmn2) localizes along filopodial actin bundles and its depletion does not affect filopodia initiation or elongation. However, Fmn2 activity is required for filopodial tip adhesion maturation and the ability of filopodia to generate traction forces. Dysregulation of filopodia in Fmn2 depleted neurons leads to compromised growth cone motility. Additionally, in fibroblasts, Fmn2 regulates ventral stress fiber assembly and affects the stability of focal adhesions. In the developing spinal cord, Fmn2 activity is required cell autonomously for the outgrowth and pathfinding of spinal commissural neurons. Our results reveal an unanticipated function for Fmn2 in neural development. Fmn2 regulates structurally diverse bundled actin structures, parallel filopodial bundles in growth cones and anti-parallel stress fibers in fibroblasts, in turn modulating the stability of substrate adhesions. We propose Fmn2 as a mediator of actin bundle integrity enabling efficient force transmission to the adhesion sites.