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Recently, Dong Chunyang, a direct doctoral graduate from the University of California, Davis and currently doing postdoctoral research at Stanford University, co-developed a new type of genetically encoded fluorescent probe, providing an innovative tool for the study of neuropeptide dynamics.

Photo | Dong Chunyang (Source: Dong Chunyang)

According to the introduction,The core idea of ​​this study is to fuse fluorescent protein with opioid receptors and reflect the binding process of opioid peptides in real time through changes in fluorescence signals caused by changes in receptor conformation.

During this period, Dong Chunyang and others designed corresponding fluorescent probes κLight, δLight and μLight for the three main opioid receptor subtypes: κ receptor, δ receptor and μ receptor.

This not only makes it possible to distinguish different types of opioid peptides, but also enables signal detection with high spatiotemporal resolution at the single cell level.

(Source: Nature Neuroscience)

Through the development and application of new fluorescent probes, this study aims to deeply explore the functions and regulatory mechanisms of the opioid peptide system from multiple levels.

This will not only help people understand the role of the opioid system in emotional and motivated behaviors, but may also provide new insights into understanding and treating related mental illnesses.

By studying receptor-ligand interactions at the molecular level, signal transduction at the cellular and circuit levels, and functional regulation at the overall behavioral level, this achievement is expected to bring revolutionary progress to the study of the opioid system.

It is an important step towards a better understanding of the endogenous opioid system and provides a valuable new tool for the study of the opioid peptide system.

Probes for both endogenous and synthetic opioids would be a great addition to the current neuroscience toolbox, providing better spatiotemporal sampling of opioid signaling.

This will not only promote the development of basic neuroscience research, but also open up new prospects for clinical applications such as pain management and addiction treatment.

(Source: Nature Neuroscience)

Starting from the “status” of opioid neuropeptides

It is reported that in the field of neuroscience, the opioid neuropeptide system occupies a central position.

As a key class of neuromodulators, opioid peptides play an indispensable role in regulating multiple physiological and pathological processes, including pain perception, reward behavior, emotional responses, and addiction.

However, in-depth research on the endogenous opioid peptide system has long been limited by technical means.

Although traditional research methods such as immunohistochemistry and radioligand binding experiments can provide static opioid peptide distribution information, it is difficult to capture the dynamic release process and fine spatiotemporal distribution patterns of opioid peptides in the living nervous system.

In particular, existing technologies often seem inadequate when exploring the functions of opioid peptides in specific neural circuits and the dynamic changes in opioid peptide release under complex behavioral states.

In order to break through the above bottleneck, Dong Chunyang and his collaborators developed this opioid receptor fluorescent probe strategy based on genetic coding.

Previously, Dong Chunyang's doctoral supervisor had developed a calcium ion probe GCaMP3, which can use fluorescence to analyze brain neural activity in multiple species, making an important contribution to the detection of neural activity with high temporal and spatial resolution.

It also laid a certain foundation for the optimization of calcium ion probes. Currently, the GCaMP series of calcium ion probes have been widely used in the field of neuroscience.

The above achievements of Professor Dong Chunyang laid the foundation for the development of new neurotransmitter probes.

Under the guidance of his supervisor, Dong Chunyang has participated in the research of red-shifted dopamine probes in recent years, and developed two serotonin probes and a variety of other neuropeptide probes.

At the same time, Dong Chunyang also collaborated with Professor Li Yulong's team at Peking University to publish a review paper on the development and application of neurotransmitter probes. These experiences provided him with some insights into exploring more complex opioid peptide probes.

On this basis, Dong Chunyang realized that the development of opioid peptide probes faced greater challenges than the neurotransmitter probes developed previously.

The opioid peptide system is very complex, including multiple endogenous opioid peptides and multiple receptor subtypes, which makes it particularly difficult to develop probes with high specificity.

And unlike other probes, there is no ready-made formula to follow for the development of opioid peptide probes, which means that each probe variant needs to be individually designed and verified, which greatly increases the workload and complexity.

For example, it is necessary to find the best fluorescent protein insertion site and connection sequence while maintaining the receptor function. During this period, a lot of molecular biology work is involved, including designing, constructing and screening hundreds of different variants.

During the in vitro characterization phase, the response of each probe variant to different opioid peptides and receptor subtypes needs to be carefully evaluated, including detailed determination of the probe's dynamic range, sensitivity, selectivity, and response kinetics.

Through a large number of in vitro characterization experiments, Dong Chunyang and others screened out variants with a larger dynamic range, higher sensitivity and higher specificity.

Subsequently, they carried out verification work on the specificity of the probe and demonstrated the specificity of κLight for dynorphin by using optogenetic stimulation of the BLA-NAc circuit between dynorphin knockout mice and wild-type mice.

This not only ensures that the probe maintains its specificity and sensitivity in complex neural environments, but also ensures the reliability of the probe for users when using it under physiological conditions.

(Source: Nature Neuroscience)

Countless repetitions, failures, optimizations, and repetitions

Dong Chunyang said that one of the goals of this study is to comprehensively characterize the pharmacological properties of these new probes in vitro and in ex vivo brain slices.

This includes determining the affinity, selectivity and kinetic characteristics of the probe for different endogenous opioid peptides and synthetic opioid peptides.

By comparing them with traditional radioligand binding experiments, they hope to verify whether these probes can accurately reflect the interaction between opioid peptides and receptors.

At the same time, it is also necessary to evaluate whether the expression of the probe will affect the normal function of endogenous opioid receptors in order to ensure its application value under physiological conditions.

The second goal of this study is to use these probes to explore the diffusion characteristics of opioid peptides in brain tissue.

Previously, it was believed that opioid peptides exerted their effects mainly through volume transfer, but direct evidence for their specific diffusion range and rate was lacking.

To this end, the research team designed a sophisticated photolysis experiment, releasing photosensitive opioid peptide precursors while simultaneously monitoring changes in fluorescence signals, thereby achieving real-time observation and quantitative analysis of the opioid peptide diffusion process for the first time.

Through this experiment,They not only revealed the diffusion constants of opioid peptides, but also provided important basis for understanding the spatial range of opioid peptide signals.

The third goal of this study is to determine the optimal electrical stimulation parameters that can trigger the release of endogenous opioid peptides, which is crucial for studying the function of opioid peptides in vivo.

By systematically adjusting the intensity, frequency, and duration of stimulation in brain slices and monitoring the response of fluorescent probes, the team hopes to identify the most effective stimulation pattern, thereby laying a foundation for electrophysiological and optogenetic experiments.

In experiments in living animals, they combined these fluorescent probes with optogenetics to explore the dynamics of opioid peptide release in specific neural circuits.

For example, by expressing the κLight probe in the nucleus accumbens and light-sensitive ion channels in the amygdala, it is possible to precisely control the activation of specific projections and observe the resulting endogenous dynorphin release in real time.

The benefit of this approach is that it not only provides unprecedented temporal and spatial resolution, but also reveals the spatial specificity of opioid peptide release.

In addition, the research team also carried out the following exploration: exploring the dynamic changes in opioid peptide release under complex behavioral states, such as fear conditioning and reward learning.

By conducting fiber optic photometry in freely moving animals, Dong Chunyang and others used probes to capture changes in opioid peptide signals associated with specific behaviors.

He still remembers the moment when he successfully captured the release of endogenous opioid peptides in living mice for the first time. He said: "It took us several years to optimize the probe and finally confirmed a variant in vitro that is expected to be successfully used in vivo."

In detail, they packaged the κLight plasmid into adeno-associated viruses, and after becoming familiar with the coordinates of the mouse brain regions, they began intracranial viral injections and then waited for the probe to be expressed in the mouse brain.

"After countless repetitions, failures, optimizations, and repetitions, I was really moved when I saw on the computer screen recording the light that the κLight probe began to emit bright signal peaks one after another as the fear conditioning stimulation progressed," said Dong Chunyang.

However, scientific rationality quickly pulled him back, and he had to prove that what he saw was the signal of dynorphin detected by κLight and not some other illusion.

After demonstrating the repeatability of the above phenomenon using multiple mice, the research team designed different experiments, especially optogenetic experiments using dynorphin knockout mice, to prove that in the absence of dynorphin and with the same stimulation, the probe would not produce a signal, thereby proving the accuracy and specificity of the probe.

Finally, when all the experiments pointed to a result with strong specificity and high signal-to-noise ratio, everyone was finally able to put their minds at ease.

(Source: Nature Neuroscience)

Finally, the related paper was published in Nature Neuroscience (IF 21.2) with the title "Unlocking opioid neuropeptide dynamics with genetically encoded biosensors".

Figure | Related papers (Source: Nature Neuroscience)

Dong Chunyang and Raajaram Gowrishankar of the University of Washington are co-first authors.

Professor Michael R. Bruchas of the University of Washington, Professor Matthew R. Banghart of the University of California, San Diego, and Professor Tian Lin of the Max Planck Florida Institute for Neuroscience serve as co-corresponding authors.

With this achievement, Dong Chunyang won the Toni Shippenberg Young Investigator Award from the National Institutes of Health.

In terms of application prospects:

First, it can be used in basic neuroscience research.

The opioid receptor fluorescent probe created this time will allow people to more accurately observe and measure the dynamic changes of opioid peptides in the nervous system, thereby helping to reveal the specific role of the opioid system in various neural processes, such as learning, memory, and emotion regulation.

Second, it can be used in pain research.

The opioid system plays a key role in pain regulation. Therefore, this probe is expected to be used to study the release pattern of opioid peptides in acute and chronic pain states, thereby helping to develop more effective pain management strategies.

Third, it can be used to study the mechanism of addiction.

By real-time monitoring of the activity of opioid peptides in the reward circuit, we may be able to better understand the neurobiological basis of drug addiction, thereby providing clues for the development of new treatments.

Fourth, it can be used for drug development and drug screening.

That is, this probe may be used for high-throughput screening to help identify new opioid receptor modulators, thereby assisting in the development of safer and more effective analgesics.

Fifth, it can be used in the study of mood disorders.

Given the close relationship between the opioid system and emotion regulation, this probe is expected to be used to study the neural mechanisms of emotional disorders such as depression and anxiety.

Sixth, it can be used in neuroimaging applications.

When these probes are modified, they are expected to be used in non-invasive brain imaging technology to observe the activity of the opioid system in the human brain.

Seventh, it can be used to develop neural regulation technology.

Combined with optogenetics or chemogenetics, these probes are expected to help develop more precise neuromodulation technologies for the treatment of diseases related to the opioid system.

Eighth, it can be used in behavioral neuroscience research.

That is, it is used to study the role of the opioid system in complex social behavior, decision-making and other advanced cognitive functions.

Ninth, it can be used to prevent drug abuse.

A deeper understanding of the function of the opioid system may help develop more effective substance abuse prevention strategies and educational approaches.

Tenth, it can be used for personalized medicine.

Studying the differences in the responses of opioid systems in different individuals in animal models may provide a theoretical basis for personalized pain treatment and addiction management.

In summary, these potential applications are not only expected to promote the development of basic neuroscience research, but may also have a significant impact on clinical medicine, drug development, and public health policies.

Of course, it will take many years of research and verification to go from basic research to practical application.

And in the future:

First, the performance of existing probes will be improved.

That is, to improve the specificity, sensitivity, dynamic range and kinetic characteristics of existing neuropeptide probes.

This may involve more complex protein engineering strategies, such as AI-assisted directed evolution and rational design guided by structural biology.

Secondly, fluorescent probes targeting more neuropeptides will be developed.

That is, to expand existing experience to other neuropeptide systems, neurotransmitters and neuromodulators.

Again, combine existing probes with other imaging techniques.

For example, combining neuropeptide probes with super-resolution microscopy or miniaturized two-photon microscopy will allow the observation of neuropeptide release dynamics at the subcellular level or in freely moving animals.

At the same time, with the help of multicolor imaging technology, people may be able to observe the dynamics of multiple neuropeptides or neurotransmitters at the same time, thereby revealing the interactions between them.

Finally, the application of these probes is not limited to basic research, but also has the potential to extend to the field of drug development, that is, to develop high-throughput screening platforms to assist in the discovery of new neuropsychiatric drugs.

For neuropeptide probes, it will also continue to drive neuroscience research towards higher spatiotemporal resolution, wider molecular diversity, and more complex behavioral paradigms, providing support for understanding brain function and developing new therapeutic strategies.

References:

1.Dong, C., Gowrishankar, R., Jin, Y.et al. Unlocking opioid neuropeptide dynamics with genetically encoded biosensors. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01697-1

Operation/Layout: He Chenlong

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