A molecular connectomics platform for multi-scale analysis of activity-dependent synapse development and plasticity

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Project summary/abstract Understanding brain function and plasticity requires innovative approaches for studying local (synaptic) molecular mechanisms that establish neural circuits (connectomes) underlying cognition and behavior. Here we propose a “molecular connectomics” approach that integrates cell-type-specific transcriptomic, proteomic, super-resolution structural imaging, and optogenetic functional analyses to investigate the role of local protein translation in connectome development. We will pilot our approach by studying the activity-dependent development of the retinogeniculate pathway, which links retinal ganglion cells (RGCs) with postsynaptic neurons in the dorsal lateral geniculate nucleus (dLGN) for conscious visual perception and behavior. Working with a transgenic mouse line (ET33-Cre) in which eye-specific RGCs are genetically accessible, we will quantify molecular (1), structural (2), and functional (3) synaptic changes during eye-specific connectome development. The postnatal development of eye-specific pathways is regulated by retinal activity, allowing us to use transgenic and pharmacological tools to disrupt RGC spiking and further quantify activity-dependent changes in local protein synthesis mechanisms driving eye-specific synapse development and plasticity. Molecular analyses (1) will use axon-TRAP to immunoprecipitate eye-specific synaptic mRNAs (local synaptic translatomes) for next-generation sequencing. Local mRNA abundance/diversity will be further validated using multi-round fluorescence in-situ hybridization and mRNA barcoding for spatial transcriptomic imaging analysis. We will quantify the local synaptic proteome using proximity-labeling and non-canonical amino-acid labeling techniques to tag and isolate synaptic protein networks for quantitative high-resolution mass spectrometry. Proteomes will be validated using super-resolution structural imaging methods. Structural analyses (2) will map the molecular refinement of retinogeniculate connections using two super-resolution imaging techniques: volumetric STochastic Optical Reconstruction Microscopy (STORM) and Expansion Microscopy (ExM). These methods will be used to quantify protein and mRNA distributions in large, circuit-level tissue volumes with subsynaptic resolution. Functional characterization eye-specific synapses (3) will be performed using channelrhodopsin-mediated optical stimulation of eye-specific axons with postsynaptic recording in dLGN cells. Post hoc super-resolution microscopy of recorded neurons allows for direct, correlative measurement of structure/function relationships underlying activity-dependent changes in synaptic strength. This work will establish a novel methodology – molecular connectomics – to link local mRNA translation mechanisms in subcellular compartments with connectome assembly and refinement. Transcriptomic/proteomic analyses will help identify differentially-regulated gene/protein candidates for future gain/loss-of-function experiments. Our long-term goal is the application of our platform to identifying molecular mechanisms of circuit dysfunction in animal models of neurodevelopmental disorders and mental illness.