Your worry, your Le fluorescent dopamine probe can "see"
Dopamine is an important neurotransmitter used to help cells transmit impulses. It is closely related to many neurological diseases such as Parkinson's disease, drug addiction, schizophrenia, ADHD and post-traumatic stress syndrome.
In order to better study the role of dopamine in physiological and pathological processes, researchers need to have a handy "weapon" that can monitor dopamine in real time, sensitively and specifically.
Prior to this, the Li Yulong laboratory of the School of Life Sciences, Peking University developed a series of fluorescent probes for monitoring neurotransmitters, including the first-generation dopamine probes.
On October 22, Li Yulong’s laboratory, in cooperation with New York University and the National Institutes of Health, published an online paper in the journal Nature Methods, reporting on the development of the new red fluorescent dopamine probe and the second generation green fluorescent dopamine probe Development and application results.
Green fluorescent probe makes cells glow by themselves
Dopamine is the most abundant catecholamine neurotransmitter in the brain. This brain secretion is related to physiological processes such as motor control, motivation, learning, memory, emotion, etc. It is responsible for transmitting information of excitement and happiness, and has many functions in regulating the central nervous system. Kind of physiological function.
In the 1950s, the Swedish scientist Alvid Carlson determined that dopamine is the role of the brain's information transmitter, making people realize that once the human dopamine regulatory system is impaired, Parkinson’s disease, schizophrenia, and tics often occur. Tourette’s syndrome, attention deficit hyperactivity syndrome, and the occurrence of pituitary tumors.
Based on this, Carlson developed drugs to treat such diseases and won the Nobel Prize in Physiology or Medicine in 2000.
"If scientists have a tool that can perceive changes in dopamine concentration with high temporal and spatial resolution, high specificity, and high sensitivity, it will be helpful to study the various functions of dopamine under physiological and pathological conditions, and to track in living model organisms. , The dynamic changes of dopamine signals under complex behavior patterns." Li Yulong said.
The traditional monitoring methods are mainly to sample the cerebrospinal fluid through microdialysis and biochemically monitor, and record through carbon fiber electrodes.
Each of these monitoring methods has some limitations, such as lack of sufficient time and space resolution, it is difficult to accurately reflect the true dynamic information of neurotransmitters; the specificity of monitoring specific neurotransmitters is not high enough; the monitoring methods are very harmful to organisms, etc. .
Therefore, scientists have been committed to optimizing existing methods, or developing new methods, trying to make up for shortcomings and make breakthroughs.
Since 2018, Li Yulong's laboratory has developed a series of genetically-encoded fluorescent probes, the GRAB probe series, which includes dopamine probes.
Li Yulong said that the most important point is that their new method "makes cells glow by themselves."
Previously, many biologists have solved the problem of how to "put" fluorescent proteins into cells.
As long as the specific information sequence encoding the gene is transferred into the cell, the cell itself will use the "central law" to "translate" this information sequence into a specific protein.
This protein that can be excited by visible light to produce red or green fluorescence will automatically "walk" to the cell membrane, and is also sensitive to dopamine, that is, it binds to a neurotransmitter and emits a fluorescent signal to report the signal position.
"Then you can take a photo or take a video. Wherever it is bright, there will be dopamine release." Li Yulong introduced to a reporter from Science and Technology Daily, so that the dynamics of the originally invisible neurotransmitter can be captured by fluorescence imaging. The changes are transformed into intuitive and easy-to-detect fluorescent signals for real-time monitoring, which creatively overcomes many problems such as low temporal and spatial resolution and poor molecular specificity in existing dopamine monitoring methods, and it hardly damages cells.
They used transfection, virus injection and other means to express the probe on cells, mouse brain slices or live fruit flies, zebrafish, and mice. They monitored the release of dopamine triggered by electrical stimulation of mouse brain slices, and in vivo Changes in dopamine signals related to olfactory stimulation, visual stimulation, learning and memory, and mating behavior were monitored in the brains of fruit flies, zebrafish and mice.
The upgraded version of the probe can "cooperate" in multiple colors
In the past two years, Li Yulong’s team has transformed and optimized the first-generation probe developed.
"Just like a computer chip, from the first generation to the second generation, its computing speed is getting faster and faster. Dopamine probes become more sensitive after the update, and the non-damaging or side effects brought by it are more Less." Li Yulong said.
The limitation of the first generation of dopamine probes is that they can only be monitored when the release of dopamine is large, and the monitoring also takes a long time.
But if there are more sensitive probes, it is possible to monitor the dynamics of dopamine more accurately in some fine animal behaviors in real time, such as when rewarded or punished, or when addiction or disease occurs. Variety.
For the new generation of dopamine probes, Li Yulong and others systematically studied its performance in cells, brain slices, fruit flies, and mice, and verified the specificity of the probe signal through a series of control experiments.
The new generation of probes also provides diverse monitoring possibilities.
"The probe is composed of a combination of fluorescent protein and neurotransmitter receptor. Based on the first generation of probes, we tried different kinds of changes in the important amino acids of the two parts of the protein interface, and Monitor the characteristics of the probes one by one; then permutate and combine the excellent amino acid changes to achieve a'strong combination' and achieve the optimal probe monitoring effect." Li Yulong said.
In addition, they tried to replace different types of fluorescent proteins and developed probes with different fluorescent colors.
"In our brains, there are many very complex chemical signals, and dopamine is just one of them." Li Yulong explained that in addition to pleasant dopamine, there is also adrenaline that is thought to manage stress, or regulate depression. Serotonin.
Therefore, when there is a red dopamine probe sensitive to "happiness" and a green neurotransmitter probe sensitive to depression or stress working together, people can see the difference between different neurotransmitters at the same time. Relationship, so as to better understand how the brain’s chemicals undergo coordinated changes.
The new red fluorescent dopamine probe they developed can be used together with other green fluorescent probes such as calcium ion probes and neurotransmitter probes to achieve simultaneous recording of multiple signals.
In addition, they optimized the second-generation green fluorescent dopamine probe with higher sensitivity and imaging signal-to-noise ratio.
Real-time detection of dopamine in vitro is not a dream
Before people monitor chemicals in the brain, they mostly used chemical methods, such as using reagents to monitor the chemical reactions of receptors; or using mass spectrometry to monitor molecules of specific sizes.
"The chemical reagents themselves are toxic; mass spectrometry must first ionize these molecules, and living cells simply cannot bear it." Li Yulong said, so these monitoring methods are not suitable for use in live animals.
Li Yulong's team used a new generation of dopamine probes to record the dynamic changes of dopamine in the deep brain regions of living animals when they are free to move.
Compared with the first-generation probes, the optimized second-generation green fluorescent probes have brighter fluorescence and more significant signal changes after combining with neurotransmitters.
In addition, the new red fluorescent probe expands from the previous single green fluorescence to multicolor fluorescence when monitoring dopamine.
Therefore, the two new probes can be more easily combined with other important technologies in the field of neuroscience, such as calcium imaging and optogenetics.
The new probes also provide new possibilities for the optimization of dopamine receptor-related drug screening.
Li Yulong told reporters that they have now begun to develop a new generation of fluorescent probes to make red light more red.
"Because in a living body, the probe is like a signal light or a navigation light. The redder light has a stronger penetrating power, a higher signal-to-noise ratio, and is easier to detect." Now to monitor these fluorescent signals, people have to give animals Only by adding an optical fiber to the body can dopamine information be monitored.
If you want to observe the "think" in the animal brain, and understand more realistically and accurately how its nervous system works, you need the fluorescent probe to have enough brightness, and you can directly see the red and piercings even outside the freely moving animals. Light with enough penetrating power.
"These are theoretically achievable." Li Yulong said, and this is where they are working hard.