# Real-time mapping of neuronal connectivity at a synapse resolution

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2024 · $403,806

## Abstract

Summary
Synapses, the connections between neurons, play a crucial role in determining circuit connectivity and function.
Visualizing their structure and dynamics is challenging due to the vast number of synapses and the complex
interactions between them. Prior studies have explored some aspects of synaptic plasticity, primarily focusing
on postsynaptic neurons and utilizing imaging techniques to observe dendritic spines, which are widely
recognized as key synaptic structures. Nevertheless, there remains a significant gap in our understanding of
how these changes are linked to their specific input sources, leaving the identity of these synapses largely
uncharted. To overcome this limitation, researchers have developed dual-component synapse detectors, like the
neurexin-neuroligin complex and split fluorescent proteins. However, split fluorescent proteins have limitations,
such as slow maturation and irreversible associations, hindering real-time synaptic observation. To address
these challenges, we developed a reversible fluorescent sensor technology using dimerization-dependent
fluorescent proteins (ddFPs) named SynapShot. These ddFPs emit fluorescence only when they heterodimerize,
which happens quickly and reversibly. With this technique, we successfully monitored synaptic dynamics in real
time, including synaptic formation and elimination, as indicated by changes in dendritic spine size. We also
distinguished connections from one postsynaptic neuron to multiple presynaptic neurons. In this proposal, we
will further explore the uncharted territory of synaptic dynamics in both local and long-range circuits in in vivo
setups. In the first aim, we will investigate the functional implications of synaptic labeling detected by the
SynapShot. Synaptic plasticity will be tested in single dendritic spines to demonstrate the ability of tracking
structural changes in real-time. In the second aim, we will explore inhibitory synaptic connections, especially
those involving genetically-defined interneuron cell types, using the SynapShot method. In the third aim, we will
employ synapShot to observe synaptic changes during the acquisition of a new learning task, study dendritic
integration dynamics, and examine the rules governing connections among engram neurons. In summary,
SynapShot provides a means to observe bidirectional changes in synaptic contacts both in vitro and in vivo and
can differentiate distinct populations of synapses when used in dual-colored configurations. This platform
contributes to understanding the relationship between time-dependent synaptic structure changes and various
brain functions and diseases. The successful completion of this project promises to be a valuable asset in
advancing the field of neural circuit research.

## Key facts

- **NIH application ID:** 10940679
- **Project number:** 1R01NS138176-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Hyungbae Kwon
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $403,806
- **Award type:** 1
- **Project period:** 2024-08-05 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10940679

## Citation

> US National Institutes of Health, RePORTER application 10940679, Real-time mapping of neuronal connectivity at a synapse resolution (1R01NS138176-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/10940679. Licensed CC0.

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