# Spatial and molecular determinants of fusion probability and timing

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2022 · $358,203

## Abstract

At chemical synapses, neurons communicate each other through release of neurotransmitters.
Synaptic transmission occurs in two kinetically distinct phases. Within milliseconds of an action potential,
synaptic vesicles fuse with the plasma membrane synchronously across multiple synapses. Following
synchronous fusion, synaptic vesicles continue to fuse with the plasma membrane over tens to hundreds of
milliseconds. This phase of neurotransmission is called asynchronous release, and it becomes more apparent
in pathophysiological conditions. Despite extensive research over the last 50 years, the nano-scale
organization of synaptic vesicle fusion sites is poorly understood. Where do synchronous and asynchronous
fusions take place within an active zone? Are fusion sites determined by the locations of calcium channels?
Which calcium sensors are responsible for asynchronous fusion? Is there a separate pool of vesicles for
asynchronous fusion? Or are the same pool of vesicles consumed for both phases? To address these
questions, we have developed zap-and-freeze electron microscopy to follow membrane dynamics millisecond-
by-millisecond. This technique couples electrical stimulation of neurons with high-pressure freezing to capture
rapid membrane trafficking events at mammalian central synapses with unprecedented spatial (1 nm) and
temporal (1 ms) resolution. Using this approach in combinations with advanced genetics, molecular biology,
and biochemical techniques, we will determine how fusion sites are organized at mammalian central synapses.
Defects in synaptic transmission play a causal role in neurological disorders. The proposed research aims to
understand the molecular mechanisms underlying synaptic transmission with the ultimate goal of
understanding the pathogenesis of these diseases.

## Key facts

- **NIH application ID:** 10443756
- **Project number:** 5R01NS105810-05
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Shigeki Watanabe
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $358,203
- **Award type:** 5
- **Project period:** 2018-09-30 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10443756

## Citation

> US National Institutes of Health, RePORTER application 10443756, Spatial and molecular determinants of fusion probability and timing (5R01NS105810-05). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10443756. Licensed CC0.

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