# Bioluminescent indicators for noninvasive imaging of acetylcholine release

> **NIH NIH R21** · STANFORD UNIVERSITY · 2021 · $433,663

## Abstract

ABSTRACT
 Although acetylcholine (ACh)-secreting neurons only constitute a small fraction of the total neurons in an
animal brain, they play a critical role in regulating essential brain functions. In particular, the dysregulation of
cholinergic neurons has been connected to the neurological deficits observed in Alzheimer’s disease (AD), the
most common neurological disorder of aging. However, while mouse models recapitulating the features of AD
have been established, the development of effective therapies or preventions has been hampered by our ability
to longitudinally monitor cholinergic function and correlate it to behavioral changes during disease development.
 In recent years, the development of genetically encoded fluorescent indicators for neurotransmitters has
made enormous progress in real-time imaging neurotransmitter release in live mouse, thereby enabling many
exciting discoveries relating the activity of specific neurotransmitters to various behavioral outputs. However, in
vivo fluorescent imaging in the brain suffers from the need to invasively insert illumination and recording devices,
which could easily create behavioral or functional deficits, especially when the region of interest is located deeply.
Thus, what is needed, and what does not exist, is a way to non-invasively and longitudinally observe the release
of neurotransmitters such as ACh from outside the animal.
We propose to create and validate ACh indicators that operate not via fluorescence but via bioluminescence,
in which a luciferase enzyme oxidizes a chemical substrate to produce light in a neurotransmitter-dependent
manner. No external excitation light is needed for bioluminescent imaging, and auto-bioluminescence is usually
missing in mammals, therefore sensitive imaging in deep tissues can be easily achieved with external detectors.
In our preliminary work, we demonstrated that Antares, a highly catalytic and red-emitting luciferase we
engineered, when coupled with novel substrates that we developed, was able to produce 55-fold brighter
bioluminescence in mouse brain, compared to the commonly used firefly luciferase. This state-of-the-art
luciferase-luciferin pair now opens the door to create sensitive bioluminescent reporters that function in the brain.
 We now propose to create neurotransmitter bioluminescent indicators (NeuBIs), starting with ACh as the
primary target. Specifically, the protein-based ACh indicator will be developed from the luciferase Antares, and
either (1) a bacterial ACh binding domain, or (2) muscarinic receptors. The created ACh indicators will be tested
in cultured neurons and mice to image ACh release. Once established, we envision these ACh indicators will be
widely used for non-invasive recording of cholinergic activity in mouse models of AD, and can serve as templates
for the engineering of other neurotransmitter bioluminescent indicators.

## Key facts

- **NIH application ID:** 10196839
- **Project number:** 1R21NS122055-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Michael Z. Lin
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $433,663
- **Award type:** 1
- **Project period:** 2021-05-01 → 2022-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10196839

## Citation

> US National Institutes of Health, RePORTER application 10196839, Bioluminescent indicators for noninvasive imaging of acetylcholine release (1R21NS122055-01). Retrieved via AI Analytics 2026-06-25 from https://api.ai-analytics.org/grant/nih/10196839. Licensed CC0.

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