2025-06-16 北京大学(PKU)
<関連情報>
- https://newsen.pku.edu.cn/news_events/news/research/14998.html
- https://www.science.org/doi/10.1126/science.adt7705
設計された遠赤ドーパミンセンサーを用いた動的神経化学ネットワークのin vivoマルチプレックスイメージング In vivo multiplex imaging of dynamic neurochemical networks with designed far-red dopamine sensors
Yu Zheng, Ruyi Cai, Kui Wang, Junwei Zhang, […] , and Yulong Li
Science Published:5 Jun 2025
DOI:https://doi.org/10.1126/science.adt7705
Editor’s summary
Understanding the interplay between different neuromodulators in vivo requires the development of reliable and specific sensors that can detect multiple neurochemicals in vivo simultaneously. Zheng et al. developed a genetically encoded dopamine sensor called HaloDA1.0 that works in the far-red spectrum, enabling the detection of in vivo dopamine signaling simultaneously with existing green and red sensors for other neuromodulators. The authors present a detailed characterization of HaloDA1.0 and demonstrate that the sensor is compatible with multicolor imaging in the brains of awake mice. The sensor enables the study of complex circuits involving multiple neuromodulators in awake animals. —Mattia Maroso
Structured Abstract
INTRODUCTION
Neurochemical signals, including neurotransmitters, neuromodulators, and intracellular signaling molecules, are dynamically modulated within networks that mediate various brain functions and contribute to neurological disorders. Dopamine (DA), one of the most important neuromodulators, interacts intricately with other neuromodulators such as acetylcholine (ACh) and endocannabinoids (eCBs), along with intracellular signals such as cyclic adenosine 5′-monophosphate (cAMP) and Ca2+. Decoding these networks is crucial for understanding neural mechanisms underlying behaviors and related disorders. However, current genetically encoded sensors are limited to the green and red spectra, hindering real-time simultaneous detection of multiple neurochemical signals. There is an urgent need to expand the spectral range of neuromodulator sensors to include far-red and near-infrared (NIR) wavelengths (i.e., those >650 nm).
RATIONALE
By utilizing G protein–coupled receptors (GPCRs) and circularly permutated fluorescent proteins (cpFPs), we and others have developed a series of green and red GPCR activation–based (GRAB) sensors, enabling the detection of neuromodulators in vivo with high spatiotemporal resolution. However, expanding this strategy with far-red/NIR proteins presents challenges because of the suboptimal properties of existing far-red/NIR fluorescent proteins. The combination of protein tags with rhodamine derivatives offers a promising alternative approach, providing a broad spectral range and high brightness. We integrated the cpHaloTag–chemical dye approach with the GRAB strategy, resulting in the creation of HaloDA1.0, the first single protein–based far-red chemigenetic sensor for neuromodulators. Capitalizing on its far-red wavelength, HaloDA1.0 provides opportunities for monitoring three neurochemicals simultaneously by combining with existing green and red sensors.
RESULTS
HaloDA1.0 demonstrated robust sensitivity, high specificity, subsecond response kinetics, and compatibility with a variety of far-red chemical dyes. Combining HaloDA1.0 with two other neuromodulator sensors in acute mouse brain slices, we achieved simultaneous imaging of three key neuromodulators, DA, ACh, and eCB, after electrical stimulation and pharmacological interventions. In zebrafish larvae, HaloDA1.0 enabled three-color imaging of DA, ATP, and Ca2+, revealing coordinated activation patterns during electrical shocks and acute seizure attacks. Our in vivo dye screening further established HaloDA1.0’s effective performance in living mice with a silicon-rhodamine dye. This enabled dual-color recording alongside optogenetic manipulations. Furthermore, we achieved simultaneous multicolor recording of spontaneous and behaviorally associated DA, ACh, and cAMP signals in the nucleus accumbens, unveiling distinct regulatory patterns and providing a comprehensive perspective of concurrent regulation of intracellular cAMP signaling.
CONCLUSION
To monitor multiple neurochemicals simultaneously, we developed the chemigenetic far-red DA sensor HaloDA1.0, enabling sensitive DA detection both in vitro and in vivo. This sensor demonstrates significant advantages for monitoring multiple neurochemical signals across diverse biological systems, including cultured neurons, acute mouse brain slices, zebrafish, and living mice. This strategy enhances our understanding of the complex interactions among various neurochemical signals, paving the way for deeper insights into neural circuit function and improved comprehension of neurological and psychiatric disorders.
OPEN IN VIEWER
Development and applications of a far-red dopamine sensor for simultaneous monitoring of multiple neurochemicals.
This sensor functions by modulating the equilibrium of the lactone form (L) and zwitterionic form (Z) of chemical dyes. Combining the far-red sensor with green and red sensors enables simultaneous monitoring of three neuromodulators or both neuromodulators and intracellular signals in various biological systems. Some schematics were created using BioRender.com.
Abstract
Dopamine (DA) plays a crucial role in a variety of brain functions through intricate interactions with other neuromodulators and intracellular signaling pathways. However, studying these complex networks has been hindered by the challenge of detecting multiple neurochemicals in vivo simultaneously. To overcome this limitation, we developed a single-protein chemigenetic DA sensor, HaloDA1.0, which combines a cpHaloTag–chemical dye approach with the G protein–coupled receptor activation–based (GRAB) strategy, providing high sensitivity for DA, subsecond response kinetics, and a far-red to near-infrared spectral range. When used together with existing green and red fluorescent neuromodulator sensors, calcium indicators, cyclic adenosine 5′-monophosphate sensors, and optogenetic tools, HaloDA1.0 showed high versatility for multiplex imaging in cultured neurons, brain slices, and behaving animals, facilitating in-depth studies of dynamic neurochemical networks.
