Nature Sub-Journal: Li Yulong’s team develops a new type of fluorescent probe to achieve accurate detection of dynamic changes in serotonin in vivo

Serotonin (5-HT), also known as serotonin, is an important monoamine neurotransmitter, widely distributed in the central nervous system and peripheral tissues.

5-HT in the central nervous system is involved in the regulation of various behaviors such as eating, sleep, learning and memory, emotions, and social interaction, while 5-HT in the periphery is critical to the regulation of physiological processes such as gastrointestinal peristalsis, vasoconstriction, and platelet aggregation.
important.

Disorders of the 5-HT system are closely related to mental disorders such as depression, post-traumatic stress disorder, drug abuse, and behavioral addiction.

Selective 5-HT reuptake inhibitors such as fluoxetine (prozac), as the most commonly used antidepressants in clinical practice, have dual effects of antidepressant and anti-anxiety.

This shows that the physiological role and clinical value of the 5-HT system are extremely important.

Regrettably, we still know very little about the physiological functions and mechanism of 5-HT system, which greatly limits the innovation and application of psychiatric precision targeted drugs.

5-HT can be released in multiple nuclei, there are more than 10 kinds of receptors in the body, the action time is as short as sub-second, as long as several days, and the system is intricate.

In recent years, scholars in the field have made some breakthroughs in the description of the whole brain projection of mouse 5-HTergic neurons and the activity recording of specific 5-HTergic neurons in the process of reward, punishment, and eating.

These studies provide important information for analyzing the functions of the 5-HT system.

However, many key mechanism issues remain unresolved.

For example, in different behaviors, how is 5-HT released systematically? What are the dynamic differences of 5-HT in different brain regions? What is the difference between the release of 5-HT under physiological and pathological conditions? Traditional methods for detecting 5-HT include microdialysis, fast scanning cyclic voltammetry, etc.
, but they are limited by the lack of temporal or spatial resolution, and the signal is not stable enough during long-term detection, so it is difficult to achieve Long-term sensitive detection of 5-HT in vivo dynamic changes.

Therefore, the development of 5-HT dynamic analysis tools with cell specificity and high temporal and spatial resolution will provide powerful tools for solving these important scientific problems.

On April 5, 2021, the Li Yulong Laboratory of Peking University published an online research paper titled: A genetically encoded sensor for measuring serotonin dynamics in the journal Nature Neuroscience, reporting the development and development of a new genetically encoded 5-HT fluorescent probe Successful application in a variety of model organisms.

Li Yulong's laboratory is a pioneer in the research and development of new neurotransmitter receptors.
In recent years, it has independently developed high-efficiency fluorescent probes for neurotransmitters or neuromodulators such as acetylcholine, dopamine, norepinephrine, and adenosine.

The above-mentioned probes have been widely used in the research of neural circuit function and the release mechanism of related signal molecules.

Following the research group’s strategy of applying G-protein coupled receptors (GPCR) to construct fluorescent probes in recent years, Li Yulong’s research group fused endogenous 5-HT receptors with cyclically rearranged green fluorescent protein.
Through a series of protein engineering optimization, successfully developed a new type of G-protein coupled receptor activation-dependent GRAB (GPCR-Activation-Based) 5-HT fluorescent probe GRAB5-HT1.
0.

The response amplitude of GRAB5-HT1.
0 to 5-HT fluorescence signal in neurons cultured in vitro is close to 300%, and it has a high degree of specificity and affinity for 5-HT molecules, and the reaction kinetic rate can reach sub-second level (Figure 1 ).

Figure 1: Characterization of the new 5-HT fluorescent probe.

(A) The expression of GRAB5-HT1.
0 and GRAB5-HTmut in neurons and the fluorescence signal response to 5-HT.

(B) Kinetic characteristics of GRAB5-HT1.
0 in HEK cells.

(C) The molecular specificity of GRAB5-HT1.
0 to 5-HT.

In order to explore the universal applicability of GRAB5-HT1.
0 in different model organisms, the researchers tested the function of the probe in Drosophila and mice.

GRAB5-HT1.
0 can not only detect the 5-HT release of a single neuron caused by physiological stimulation in the brain of living Drosophila, it can also be combined with the optical fiber recording system to detect the sleep-wake process in real time in freely moving mice.
The dynamic changes of 5-HT.

Interestingly, combined with the two-photon imaging system, GRAB5-HT1.
0 can also detect the changes in 5-HT levels caused by psychostimulants in mice with fixed heads for a long time (Figure 2).

Figure 2: Application of the new 5-HT probe.

(A) The expression of GRAB5-HT1.
0 in Drosophila mushroom body and its response to smell, abdominal electric shock, and fluorescence signal of exogenous 5-HT.

(B) GRAB5-HT1.
0 detects the dynamic changes of endogenous 5-HT during sleep and wakefulness in freely moving mice.

(B) GRAB5-HT1.
0 detects changes in 5-HT levels caused by psychostimulants in mice with fixed heads.

The new 5-HT fluorescent probe is an important tool to further explore the functions of the 5-HT system, laying a methodological foundation for analyzing the complex neural circuits of the brain.

At the same time, this work also further proves the universality of the research group using the principle of GPCR activation to construct a fluorescent probe strategy.

We look forward to the development of more fluorescent probes for different important signal molecules in the near future, opening up new ways for the precise analysis of important molecular functions such as neurotransmitters and neuromodulators.

Wan Jinxia, ​​a doctoral student in the School of Life Sciences, Peking University, is the first author, and Professor Li Yulong from the School of Life Sciences, Peking University is the corresponding author.

Peking University doctoral students Li Xuelin, Qian Tongrui, Zeng Jianzhi, Deng Fei, etc.
have made important contributions to the article.

This work was assisted by the cooperation of the Xu Min Laboratory of the Chinese Academy of Sciences Brain Technology Excellence Innovation Center, the Jing Miao Laboratory of the Beijing Brain Science and Brain Research Center, the University of Virginia J.
Julius Zhu and the B.
Jill Venton Laboratory, etc.
, And received strong support from Peking University State Key Laboratory of Membrane Biology, National Natural Science Foundation of China, Northern Science Center for Brain Science and Brain-like Research, Peking University-Tsinghua Life Sciences Joint Center, and the American Brain Project.

Link to the paper: Open for reprint