Experts comment on Nat Neurosci | Li Yulong's laboratory develops a new fluorescent probe to accurately detect the dynamic changes of serotonin in vivo

Comments | Ren Jing (UK MRC), Ren Chaoran (Jinan University), Li Xiaoming (Zhejiang University), editor in charge | xi 5-hydroxytryptamine (5-HT), also known as serotonin (serotonin), is an important monoamine neurotransmitter Quality, 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 (PTSD), substance 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, Peking University’s Li Yulong Laboratory published a research paper titled A genetically encoded sensor for measuring serotonin dynamics online in Nature Neuroscience, reporting the development of a new genetically encoded 5-HT fluorescent probe and its application.
Successful application in multiple 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 receptor (GPCR) to construct fluorescent probes in recent years, Li Yulong’s research group combined the endogenous 5-HT receptor with the circularly rearranged green fluorescent protein (circularly).
Permutated green fluorescent protein, cpGFP) fusion, through a series of protein engineering optimization, successfully developed a new 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 helped 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, Peking University-Tsinghua Life Sciences Joint Center, and the American Brain Project.

Editor's postscript: On February 25, 2021, BioArt reported (Nat Methods | New method can visually detect changes in serotonin under physiological conditions) An article published by the University of Virginia in the journal Nature Methods A fast, high-affinity fluorescent serotonin biosensor engineered from a tick lipocalin, reported that a new gene probe can be used to detect the dynamic changes of serotonin (serotonin) under the natural physiological activities of organisms.

Then, shortly after the article was published, the previous preprint of the article was nearly withdrawn, and some explanations were made, indicating that the relevant experimental results cannot be repeated in the laboratory (see the figure below; /10.
1101/2020.
04.
18.
048397v2).

Original link: https://doi.
org/10.
1038/s41593-021-00823-7 Expert comment Jing Ren (Group leader at MRC Laboratory of Molecular Biology, Cambridge, UK) Wan et al.
presents a novel genetically encoded neurotransmitter sensor that can be used to image serotonin (5-HT) release, allowing neuroscientists to study the precise mechanisms of 5-HT signaling.
Mental illnesses affect more than 1 billion people globally.
The serotonin (5-HT) system is the most frequently targeted neural system for treating mental illnesses, including depression and anxiety.
Thus, understanding the 5-HT system is crucial for both basic and translational studies.
However, the 5-HT system is extremely complicated across anatomical, physiological and behavioral levels, making it one of the most mysterious neural systems.
It has brought enormous challenges to the study of its function and signaling mechanism.
In order to understand when, where and how 5-HT is released during normal daily life and mental illness conditions, we need to “see” 5-HT in real- time and we need tools like genetically encoded neurotransmitter sensors to help us “see” it.
Genetically encoded neurotransmitter sensors are generally assembled from two components, a transmitter sensing domain and a reporter domain (eg fluorescent protein).
This GRAB5-HT1.
0 sensor developed in Prof.
Yulong Li's lab is one of their brilliant designs utilizing G-protein coupled receptors (GPCRs) as the sensing domains.
It is based on the serotonin receptor HTR2C.
GRAB5-HT1.
0 was shown to detect serotonin in cultured cells, mouse brain slices, living fly and mouse brains.
It is highly specific,exhibiting excellent sensitivity for measuring extracellular 5-HT dynamics.
By using this sensor, Wan et al.
finds that 5-HT levels change dynamically throughout the sleep–wake cycle in mice, indicating the wide applications for GRAB5-HT1.
0 sensor in imaging 5-HT changes in the brains of live animals.
We expect that with innovative tools like GRAB5-HT1.
0 and its upgrading versions, the relationships among the activity of 5-HT neurons, the release of 5-HT and its influences on the downstream neurons will be revealed soon.
Hopefully, in the near future, these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (UK MRC Molecular Biology Laboratory team leader) Li Yulong's team has developed a new genetically encoded 5-HT fluorescent probe, which enables neuroscientists to accurately study the 5-HT signaling pathway.
Wan et al.
finds that 5-HT levels change dynamically throughout the sleep–wake cycle in mice, indicating the wide applications for GRAB5-HT1.
0 sensor in imaging 5-HT changes in the brains of live animals.
We expect that with innovative tools like GRAB5-HT1.
0 and its upgrading versions, the relationships among the activity of 5-HT neurons, the release of 5-HT and its influences on the downstream neurons will be revealed soon.
Hopefully, in the near future, these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (the leader of the research group of the British MRC Molecular Biology Laboratory) Li Yulong's team has developed a new type of heritable Encoded 5-HT fluorescent probe, which enables neuroscientists to accurately study the 5-HT signaling pathway.
Wan et al.
finds that 5-HT levels change dynamically throughout the sleep–wake cycle in mice, indicating the wide applications for GRAB5-HT1.
0 sensor in imaging 5-HT changes in the brains of live animals.
We expect that with innovative tools like GRAB5-HT1.
0 and its upgrading versions, the relationships among the activity of 5-HT neurons, the release of 5-HT and its influences on the downstream neurons will be revealed soon.
Hopefully, in the near future, these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (the leader of the research group of the British MRC Molecular Biology Laboratory) Li Yulong's team has developed a new type of heritable Encoded 5-HT fluorescent probe, which enables neuroscientists to accurately study the 5-HT signaling pathway.
0 sensor in imaging 5-HT changes in the brains of live animals.
We expect that with innovative tools like GRAB5-HT1.
0 and its upgrading versions, the relationships among the activity of 5-HT neurons, the release of 5-HT and its influences on the downstream neurons will be revealed soon.
Hopefully, in the near future, these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (UK MRC Molecular Biology Laboratory Research Group Leader) Li Yulong's team has developed a new genetically encoded 5-HT fluorescent probe that enables neuroscientists to accurately study 5-HT signaling pathways.
0 sensor in imaging 5-HT changes in the brains of live animals.
We expect that with innovative tools like GRAB5-HT1.
0 and its upgrading versions, the relationships among the activity of 5-HT neurons, the release of 5-HT and its influences on the downstream neurons will be revealed soon.
Hopefully, in the near future, these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (UK MRC Molecular Biology Laboratory Research Group Leader) Li Yulong's team has developed a new genetically encoded 5-HT fluorescent probe that enables neuroscientists to accurately study 5-HT signaling pathways.
these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (Leader of the MRC Molecular Biology Laboratory in the UK) and Li Yulong's team developed a new type A genetically encoded 5-HT fluorescent probe that enables neuroscientists to accurately study the 5-HT signaling pathway.
these findings will help us to tackle the 5-HT system and we can finally understand the role of serotonin in the aetiology of mental illnesses.
Ren Jing (Leader of the MRC Molecular Biology Laboratory in the UK) and Li Yulong's team developed a new type A genetically encoded 5-HT fluorescent probe that enables neuroscientists to accurately study the 5-HT signaling pathway.

More than 1 billion people worldwide are suffering from mental illnesses, and the 5-HT energy system is a therapeutic target for many mental illnesses, including depression and anxiety.

Therefore, a better understanding of the 5-HT energy system is essential for both basic research and translational medicine research.

However, the 5-HT energy system is very complex both in anatomical structure and at the level of physiological behavior, which makes it one of the most mysterious nervous systems.

This also brings great challenges to the research of 5-HT energy system functions and signal pathways.

In order to understand when, where and how 5-HT is released under physiological and pathological conditions, we need to observe the dynamic changes of 5-HT in real time, and genetically-encoded neurotransmitter probes can help us better Ground observation of 5-HT.

The genetically encoded neurotransmitter fluorescent probe usually includes two parts, namely, a neurotransmitter binding domain and a fluorescent protein reporter domain.

The 5-HT probe GRAB5-HT1.
0 developed by Professor Li Yulong's laboratory is one of their masterpieces of using G protein-coupled receptor (GPCR) as a neurotransmitter binding domain to develop probes.

The neurotransmitter binding domain of the GRAB5-HT1.
0 probe is the 5-HT receptor HTR2C.

The GRAB5-HT1.
0 probe can detect the dynamic changes of 5-HT in cultured cells, mouse brain slices, living fruit flies and mouse brains.

GRAB5-HT1.
0 probe also has high specificity, and also shows high sensitivity when detecting the dynamic changes of extracellular 5-HT.

In addition, Li Yulong's team used this probe to detect the dynamic changes of 5-HT during the sleep-wake cycle of freely moving mice, indicating that the GRAB5-HT1.
0 probe can be widely used to detect 5-HT in the brains of living animals .

We hope to use innovative tools such as the GRAB5-HT1.
0 probe and its upgraded version to reveal the relationship between 5-HT neuronal activity and 5-HT release and its impact on downstream neurons as soon as possible.

I hope that in the near future, these new discoveries will help us better understand the 5-HT energy system and ultimately understand the role of 5-HT in mental illness.

Expert comment Ren Chaoran (Researcher of Jinan University, winner of National Excellent Youth) 5-HT is an important monoamine neurotransmitter in the brain, which can act on 5-HT receptors expressed on cells in different brain regions to regulate neural activity , Which in turn affects the body's important physiological functions such as emotion, cognition, and sleep.

In the brain, 5-HT is mainly synthesized by 5-HTergic neurons in the dorsal raphe nucleus (DRN), which are widely innervated in many downstream brain regions and constitute the 5-HT system of the central nervous system.

The structure of the 5-HT system is complex.
On the one hand, 5-HT neurons project extensively.
On the other hand, a single brain area often contains many different types of receptors and neurons.

Therefore, analyzing the role and regulation mechanism of 5-HT system in specific behaviors has always been one of the frontier hotspots of neuroscience research.

The precise measurement of the dynamic changes of 5-HT levels in specific brain regions is the key to answering the above questions.

The team led by Professor Li Yulong of Peking University is a pioneer in the research and development of new neurotransmitter receptors.
In recent years, they have independently developed probes that can efficiently detect neurotransmitters such as acetylcholine, dopamine, norepinephrine, and adenosine.
Acupuncture has been widely used in the research of neural circuit function and the mechanism of transmitter release regulation.

Recently, Nature Neuroscience reported a new 5-HT probe, 5-HT1.
0 independently developed by Professor Li Yulong’s team.

By modifying the natural 5-HT receptor 5-HT2C, they cleverly inserted the conformation-sensitive green fluorescent protein cpGFP into it, so that the chimera can undergo conformational changes when it binds to 5-HT, and the green fluorescent signal will rise.
Therefore, changes in 5-HT levels can be reflected by detecting changes in fluorescence brightness.

In this work, they also conducted a series of in vitro (HEK293T cells, mouse brain slices) and in vivo (Drosophila, mouse) experiments to prove that the 5-HT1.
0 probe has high affinity and specificity for 5-HT.
It has the advantages of sex, rapid response, etc.
, and the expression of the probe will not significantly interfere with the intracellular signal pathway.

More importantly, the 5-HT1.
0 probe combined with genetics (such as viral vectors) and in-vivo optical recording tools (such as optical fiber recording) can dynamically observe different types of neurons in specific brain regions in freely moving animals 5-HT input received.

The development of the 5-HT1.
0 probe undoubtedly provides a great tool for analyzing the role and regulation mechanism of the 5-HT system in specific behaviors.
It is also expected that Professor Li Yulong's team will make further achievements in the field of neurotransmitter probe research and development.

Expert comment Li Xiaoming (Professor of Zhejiang University, Changjiang Scholar) In the 1980s, people discovered that 5-HT reuptake inhibitors have antidepressant effects, which triggered an upsurge in 5-HT system research.

In the following thirty years, people conducted a more comprehensive study on the 5-HT system.

These studies describe the complexity of the 5-HT receptor system and also reveal the important regulatory role of the 5-HT system in emotion, homeostasis, and instinctive behavior.

However, due to the lack of accurate 5-HT detection methods, the research progress of 5-HT signaling is very slow; how does the dynamic change of 5-HT regulate physiological functions, and whether the delay of the onset time of 5-HT reuptake inhibitors is related to the dynamics? The change is related and it is still unclear.

In recent years, the team of Professor Li Yulong of Peking University has been committed to the development of genetically encoded neurotransmitter/modulatory fluorescent probes, and creatively realized a variety of neurotransmitter/modulatory (such as acetylcholine, dopamine, norepinephrine, adenosine) Etc.
) In-vivo visual detection at high spatial and temporal resolution has promoted neurotransmitter research into a new era of in-vivo quantitative research.

Using these probes, people have a new understanding of the functions of neurotransmitters and modulators such as dopamine and adenosine.

In the report and academic posters of the 2019 Cold Spring Harbor Asia Conference, Professor Li Yulong's team initially demonstrated the research progress of 5-HT fluorescent probes, which is very exciting.

Recently, the new 5-HT probe developed by Professor Li Yulong’s team was officially published in Nature Neuroscience.

The probe has high specificity, suitable affinity and dynamic characteristics, and can be used to detect the dynamic changes of 5-HT in the brain under physical and pathological conditions.

The data of the two common model animals of fruit flies and mice in the article indicate that the probe has high signal-to-noise ratio and spatiotemporal resolution in in vivo applications, and can reflect the dynamic changes of 5-HT levels in different brain regions over time .

My laboratory was fortunate to use the 5-HT probe of Professor Li Yulong’s team in advance to detect the dynamic changes of 5-HT in specific brain areas in the mouse brain under specific behavioral conditions, and found that it has extremely high sensitivity.

It is believed that this probe will bring breakthrough progress in the research of 5-HT system.

In addition, in recent years, in the research and development of acetylcholine and dopamine fluorescent probes, Professor Li Yulong’s team has continued to update and iterate, and developed high-sensitivity probes that can be applied to different brain regions.

Compared with the acetylcholine and dopamine system, the 5-HT system is more complex and has a wider range of action.

Therefore, we are looking forward to the emergence of next-generation 5-HT probes.

We were shocked by the series of work done by Professor Li Yulong’s team on neurotransmitter/modulated fluorescent probes, and we are very grateful for their openness and sharing of material resources.

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