SynActive’ – a genetic toolbox to study the connectome and proteome of learning and memory-associated synapses

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.