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.
By combining advanced methods in transcriptomics, proteomics, super-resolution structural imaging, and functional recording methodologies we propose a quantitative, multi-scale approach for investigating the mechanisms that regulate local protein translation during synaptic competition and refinement in genetically- targeted visual circuits for conscious visual perception and behavior.
