# Development of multi-color, bright chemigenetic indicators to image synaptic transmission

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA AT DAVIS · 2020 · $471,000

## Abstract

Abstract
Activity of the brain across structures is an orchestrated process that spans a broad range of time
and space scales. Highly coordinated communication is what activate responses to stimuli, makes
behavior possible, and generates memories. To observe exchanges of information at the cellular
and synaptic level, neuroscientists have been increasingly using non-invasive imaging techniques
that rely on genetically encoded indicators based on fluorescent proteins (FPs). Calcium sensors,
such as GCaMPs, are routinely used to probe neuronal firing, and, more recently, indicators
targeting small molecule neurotransmitters/modulators (e.g.: glutamate, GABA, dopamine) were
developed and quickly gained popularity in the field. Small-molecules indicators allow direct
visualization of chemical communication, providing a large amount of information on the type of
inputs used in neuronal networks in association with stimuli. Sensors based on fluorescent
proteins are suitable for imaging fast, transient synaptic responses, however they are not able to
provide information on integrated signals from large scale areas in the brain. Here, we propose a
new sensor design for probing small molecules in the brain. We take advantage of chemical
fluorophore, which are brighter, more photostable and have broader color-spectrum compared to
FPs. The development of various in-cell labeling strategies have put the chemical fluorophore
under genetic control. We thus propose to develop chemigenetic sensors based on self-labeling
proteins (SNAP-tag, Halo-tag), which is engineered to become dependent on the presence of a
neurotransmitter/modulator. In a preliminary study, we coupled a split version of SNAP-tag to a
glutamate-binding protein (GltI, from E. coli) and showed that labeling of the construct with a
fluorescent dye occurs proportionally to the amount of glutamate in solution. Aim 1 will build upon
our preliminary results to improve the design of the construct and optimize the dynamic range of
the sensor. We will use rational engineering, as well as random mutagenesis combined with high-
throughput screening to improve the current design. We will then proceed with characterization
of the sensor in vitro, as well as ex vivo in HEK cells and dissociated neurons. Ultimately, we will
perform testing in cultured and acute hippocampal slices with 2-photon microscopy. Aim 2 will
expand the scope of the sensor by exploring a broader range of fluorescent dyes and color
variants to probe into the multiplexing capabilities of the sensor. Furthermore, we will incorporate
the modular design into binding proteins derived from sensors for other
neurotransmitters/modulators, with particular attention to GABA, acetylcholine and serotonin. We
will also explore the use of Halo-tag as a self-labeling protein, to increase the multiplexing ability
of our approach to more than one type of input signal.

## Key facts

- **NIH application ID:** 10048014
- **Project number:** 1R21EY031858-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA AT DAVIS
- **Principal Investigator:** Lin Tian
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $471,000
- **Award type:** 1
- **Project period:** 2020-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10048014

## Citation

> US National Institutes of Health, RePORTER application 10048014, Development of multi-color, bright chemigenetic indicators to image synaptic transmission (1R21EY031858-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10048014. Licensed CC0.

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