Experts comment on Nat Biotechnol's Li Yulong laboratory to develop a new fluorescent probe for detecting the spatiotemporal dynamics of endocannabinoids

Comments | Liu Zhijie (Shanghai University of Science and Technology), Li Xiaoming (Zhejiang University), Pablo Castillo (Albert Einstein College of Medicine), editors | xi In human history, cannabis has been domesticated and used for thousands of years
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Completed in the Han Dynasty of China's oldest medicine book "Shen Nong's Herbal Classic" reads: "Ma Fen (ie marijuana fruit), spicy level
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Main debility, Lee five internal organs, blood, cold
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More food, it is Hell, go crazy
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Take a long time
, understand the gods, and be light.
” This not only introduces the medicinal effects of cannabis, but also describes the changes in mental activities such as hallucinations caused by people ingesting cannabis
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It is now known that the effect of cannabis recorded in ancient books on human mental activity is realized by the effect of its natural component cannabinoid molecules into the human body and acting on the endocannabinoid system
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Figure 1: The morphology of cannabis shows the endocannabinoids (endocannabinoids, eCBs), including arachidonic acid glyceride (2-Arachidonoylgylcerol, 2-AG) and N-arachidonoylethanolamide (AEA), which are produced by A class of lipid neuromodulator molecules synthesized and released by neurons
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Classical neurotransmitters are released from the presynaptic terminals to activate postsynaptic membrane receptors; while eCB "does the opposite"-released at postsynaptic neurons, acting retrogradely on specific types of neuron synapses Cannabinoid receptor CB1R (Cannabinoid receptor type 1) plays a presynaptic inhibitory effect
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Studies in the past two decades have found that eCB participates in the regulation of synaptic plasticity in multiple brain areas of the brain, including the cortex, hippocampus, striatum, amygdala, hypothalamus and substantia nigra, and is essential for maintaining the normal function of the nervous system.
It is important and is closely related to a variety of physiological processes such as rewarding behavior, energy metabolism, learning and memory, sleep and awakening, and emotions
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Abnormal regulation of the endocannabinoid system is also closely related to neurodegenerative diseases, epilepsy, addiction, depression and schizophrenia, and many other neurological and mental diseases
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However, the lack of high-sensitivity, high-temporal-resolution experimental methods to directly detect the dynamic changes of eCB in vivo has greatly restricted people's research on its important functions and molecular regulation mechanisms under physiological and pathological conditions
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On November 11, 2021, Peking University's Li Yulong Laboratory published a research paper entitled A fluorescent sensor for spatiotemporally resolved imaging of endocannabinoid dynamics in vivo in Nature Biotechnology, reporting a new gene-encoded endocannabinoid probe GRABeCB2 .
0 development and application in a variety of scenarios inside and outside the body
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Since 2018, Li Yulong’s laboratory has developed fluorescent probes for neurotransmitters/modulators such as acetylcholine, dopamine, norepinephrine, adenosine, serotonin, etc.
The release of GRABeCB2.
0 this time is another masterpiece.
Further expand the GRAB series of fluorescent probe family
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In this work, the Li Yulong laboratory used its previously designed GRAB probe strategy (GPCR activation-based sensor) to develop the eCB probe eCB2 based on the human cannabinoid receptor CB1R and the cyclically rearranged green fluorescent protein cpEGFP.
0
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In HEK293T cells and primary neurons cultured in vitro, the eCB2.
0 probe showed good cell membrane localization, and the exogenously added cannabinoids AEA and 2-AG had submicromolar to micromolar affinity.
Second-level kinetic response and high molecular specificity (Figure 2)
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In addition, the eCB2.
0 probe does not activate the downstream signaling pathways of GPCRs, indicating that the probe itself has no significant effect on the normal physiological activities of cells
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Figure 2: The performance of eCB2.
0 on HEK293T cells and primary neurons.
So, can eCB2.
0 be used to detect the eCB released by neurons? The author started with primary neurons cultured in vitro and proved that eCB2.
0 can detect the release of eCB from neurons induced by electrical stimulation
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Through pharmacological methods, the author found that the type of eCB released by neurons at this time was mainly 2-AG
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In addition, the author also found that even without electrical stimulation, spontaneous eCB2.
0 signals with a diameter of about 10 microns in discrete distribution can be recorded, indicating that the release of eCB has specific and local characteristics
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In the acute brain slice, which is closer to physiological conditions, both electrical stimulation and high potassium solution stimulation can cause the release of eCB, and its local release characteristics have also been verified again: eCB2.
0 can bind with a single axon (bouton ), that is, the spatial resolution of a single synapse, the eCB signal on CB1R-positive neurons in the hippocampus is detected
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The basolateral amygdala (BLA) is a key brain area that mediates fear responses and processes aversion memories
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The endocannabinoid receptor CB1R has high levels of expression here
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In order to study the dynamic changes of eCB in the BLA brain area of ​​live animals when they are stimulated by injury, the author used AAV virus to express eCB2.
0 probe in mouse BLA and used optical fiber recording to successfully detect the BLA when the mouse foot was given an electric shock.
ECB signal in brain area
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On the other hand, in the CA1 brain area of ​​the hippocampus, the author used in vivo two-photon two-color microscopy to record the calcium signal and eCB signal of the neurons in the CA1 area of ​​the mouse when running, and found that the running behavior is always accompanied by the CA1 neurons The excitement and the increase of eCB signal indicate that the eCB2.
0 probe can detect the dynamic changes of endocannabinoids in the brain of mice under physiological conditions (during exercise) in real time
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When the brain is in a disease state, what changes will the release of eCB show? Past studies have found that animals with compromised endocannabinoid systems are prone to epilepsy
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In order to explore the connection between epilepsy and eCB signaling, the authors constructed epilepsy model mice and recorded the neuronal activity and eCB dynamics during epileptic seizures in mice with the help of two-photon imaging
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Interestingly, when mice were induced to develop epilepsy, neurons in the CA1 region of the hippocampus showed violent calcium signal activity and accompanied by a strong increase in eCB signals; more interestingly, immediately after the epileptic seizure, the author was in the CA1 brain The strong "calcium wave" signal and "eCB wave" signal transmitted along the horizontal direction were recorded in the area (see video)
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Video: Changes in endocannabinoid signals and calcium signals in the hippocampus CA1 brain area of ​​mice before and after epileptic seizures.
Pay attention to the transmission of endocannabinoid waves and calcium waves.
The high-temporal and spatial resolution records of the sci-fi provide a powerful new tool for the scientific community to deeply study the important functions and regulation mechanisms of endocannabinoids under physiological and pathological conditions
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It is worth mentioning that the collaborator of this research, the Ivan Soltesz laboratory of Stanford University in the United States, used eCB2.
0 probes to conduct further research on the dynamics of endocannabinoids in the brain and their role in epilepsy.
The results were published in Neuron magazine at the same time
.

Figure 5: Ivan Soltesz laboratory analyzed the molecular characteristics and spatiotemporal dynamics of cannabinoid signals in the hippocampus of living animals with the help of eCB2.
0 probes
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They found that compared with normal physiological activities, epilepsy caused the release of a large amount of 2-AG, and 2-AG provided the basic substrate for long-term stroke-like symptoms (Farrell et al, 2021)
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Dong Ao, a 2021 PhD graduate of Peking University Joint Center for Life Sciences, is the first author of this article, and Professor Li Yulong from the School of Life Sciences of Peking University is the corresponding author
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Tsinghua University undergraduate graduate He Kaikai, Peking University doctoral students Cai Ruyi and Wang Huan, undergraduate graduate Duan Jiali, etc.
made important contributions to the article
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This work was coordinated by the National Institutes of Health David Lovinger Laboratory, Cold Spring Harbor Laboratory Bo Li Laboratory, Stanford University Jun Ding Laboratory and Ivan Soltesz Laboratory and other teams, and the National Key Laboratory of Membrane Biology, Peking University Laboratory, Peking University-Tsinghua Life Science Joint Center
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For more details about the work of Li Yulong’s laboratory, please visit: http://yulonglilab.
org/ Expert Comment Liu Zhijie (Professor of Shanghai University of Science and Technology, Dean of Dadao Academy, Executive Director of iHuman Institute) Endocannabinoid System (ECS) is widely distributed in The nervous system, composed of endocannabinoids and their receptors, and enzymes responsible for the synthesis and degradation of endocannabinoids, plays a vital role in all stages of human development to adulthood
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In addition to the main cannabinoid receptors CB1 and CB2, research in recent years has also continued to discover new cannabinoid targets that play a role in the nervous system, immune system, and cardiovascular system
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ECS is closely related to pain, obesity, addiction, inflammation, and other diseases, so the major components of the system are also potential drug targets
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Research on ECS is inseparable from suitable tools
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However, limited by the lack of technology, we were unable to directly detect endocannabinoids in real time in the past, especially to study the relationship between the dynamics of endocannabinoids and behavior in vivo
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Professor Li Yulong has focused on the development of GPCR-based fluorescent probes for many years, and has established a GRAB probe library with rich members, enabling the detection of a variety of neurotransmitters and neuromodulator molecules
.

In this new study, the GRAB-eCB2.
0 probe showed good performance, and stable endocannabinoid signals were detected on cells cultured in vitro, brain slices, and live mice, which will be useful for future analysis of endogenous cannabinoids.
The function of sex cannabinoids in the body opens up a whole new way
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It is expected that they will develop more innovative probes in the future
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Expert comment Li Xiaoming (Professor of Zhejiang University, Changjiang Scholar) China is the earliest country to cultivate and use cannabis.
The early medicinal books "Shen Nong's Materia Medica" and "Compendium of Materia Medica" contain a large number of records on the medical and health care uses of cannabis, which can be used for treatment of pain, seizures, vomiting and so on
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In addition to the naturally occurring cannabinoids, studies have found that there are also synthetic and secreted cannabinoid substances in the body, called endocannabinoids (eCB), which can regulate mood, sleep, appetite and other neural activities
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Whether it is the tetrahydrocannabinol in the plant cannabis or the endocannabinoid eCB, they all realize signal transmission by acting on the cannabinoid receptors CB1 and CB2 in the human body, which together constitute the endocannabinoid system
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Different from neurotransmitters in the traditional sense, the endocannabinoid eCB is not synthesized and stored in secretory vesicles.
The synthesis of eCB is an "on-demand" method, which is only synthesized under conditions that require release.
It is immediately released between the synapses, and then quickly inactivated by reuptake or enzymatic degradation
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Although we have a better understanding of the biochemical and physiological characteristics of eCB, due to technical limitations, the spatiotemporal dynamics of when, where, and how eCB is released under physiological and pathological conditions are still unclear.
The research and understanding of functions and information pathways have brought great challenges
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Recently, Nature Biotechnology reported on the new eCB probe-RABeCB2.
0 independently developed by the team of Professor Li Yulong of Peking University
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The principle of this new probe is to use the human cannabinoid receptor CB1 as the backbone of the probe, and insert the green fluorescent protein (cpEGFP), which is sensitive to structural changes, into the receptor.
The modified CB1 binds to eCB.
The conformational change is triggered, and the conformational change is converted into a fluorescent signal, so the change in eCB level can be reflected by detecting the change in fluorescence brightness
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In cells cultured in vitro and acute mouse brain slices, GRABeCB2.
0 has been proven to have the advantages of strong affinity, high sensitivity, molecular specificity, and fast response speed
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Importantly, combined with experiments such as eCB probes, genetics, and in-vivo fiber recording, GRABeCB2.
0 can also monitor the amygdala and hippocampus eCB in the brain of mice after plantar electric shock, exercise and epilepsy in real time.
The level changes indicate that the GRABeCB2.
0 probe can be effectively used to monitor the "every move" of eCB in the brains of living animals
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Professor Li Yulong returned to China in 2012 and established his own laboratory in the School of Life Sciences, Peking University.
The team he leads is a pioneer in the research and development of new neurotransmitter receptors
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In the past few years, we have focused on synapses, the basic structure of neuronal communication.
On the one hand, we have devoted ourselves to the development of new genetically-encoded fluorescent probes to analyze the complex functions of the nervous system on time and space scales
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On the other hand, it uses advanced tools to analyze the communication connections of neurons under physiological and pathological conditions
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In recent years, they have independently developed the ability to detect acetylcholine (Nature Methods, 2020; Nature Biotechnology, 2018), dopamine (Nature Methods, 2020; Cell, 2018), norepinephrine (Neuron, 2019), Adenosine (Science, 2020), 5-hydroxytryptamine (Nature Neuroscience, 2021) and other neurotransmitter probes.
The above-mentioned probes have been widely used in the research of neural circuit function and the mechanism of transmitter release regulation, etc.
, to promote neurotransmitter Qualitative "dark matter" research has entered a new era of "visualization" research
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Expert reviews Pablo Castillo (Albert Einstein College of Medicine, Harold and Muriel Block Chair in Neuroscience)Seeing is believing.
Imagine a molecular sensor that allows you to visualize and characterize the release of an endogenous ligand with a superb spatiotemporal resolution and excellent sensitivity.
Based on on G protein-coupled receptors (GPCRs) conjugated with a fluorescent protein, Yulong Li and collaborators have successfully developed fluorescent sensors for visualizing the release of several neurotransmitters and neuromodulators in the brain.
In a new study published in Nature Biotechnology (Dong et al, 2021), they report a new GPCR activation-based (GRAB) sensor for endocannabinoids (eCBs), GRAB eCB2.
0, which can be used to characterize eCB release both in vitro and in vivo.
Thus far,eCB release has been indirectly estimated by the functional consequences of eCB binding to cannabinoid receptors.
Now, thanks to this new sensor, we can visualize eCB release and address mechanistic questions that will significantly advance our understanding of eCB signaling in the normal brain and in several brain disorders.
As the saying goes: seeing is believing
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The imaging of molecular probes can detect and characterize the release of specific endogenous ligands with ultra-high temporal and spatial resolution and excellent sensitivity
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Based on G protein-coupled receptors and fluorescent proteins, Li Yulong and his collaborators have successfully developed several fluorescent probes to detect the release of neurotransmitters and neuromodulators in the brain in the past
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In this latest work published in Nature Biotechnology (Dong et al, 2021), the authors report a new type of G protein-coupled receptor activated (GRAB) probe eCB2.
0 for research in vitro and in vivo In case of release of endocannabinoids
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Prior to this, the release of endocannabinoids was indirectly indicated by detecting the functional consequences of endocannabinoid binding to cannabinoid receptors
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Now, thanks to this new probe, we can directly "see" endocannabinoids and try to solve some mechanism problems, helping us greatly deepen our understanding of the role of endocannabinoid signals in normal brain and brain diseases.

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Link to the original text: https://doi.
org/10.
1038/s41587-021-01074-4.
Plate maker: Notes for reprinting on the 11th [Non-original article] The copyright of this article belongs to the author of the article.
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Reprinting is prohibited without permission.
The author has all legal rights, and offenders must be investigated
.