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

> **NIH NIH DP2** · UNIV OF MARYLAND, COLLEGE PARK · 2020 · $2,239,818

## Abstract

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...

## Key facts

- **NIH application ID:** 10002769
- **Project number:** 1DP2MH125812-01
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** Colenso McNaughton Speer
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $2,239,818
- **Award type:** 1
- **Project period:** 2020-09-01 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10002769

## Citation

> US National Institutes of Health, RePORTER application 10002769, A molecular connectomics platform for multi-scale analysis of activity-dependent synapse development and plasticity (1DP2MH125812-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10002769. Licensed CC0.

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