Dopamine fluorescent probe to map the brain's "roading light"

A thick banana scent attacked a fruit fly in the dark, it "buzzed" excited, flapping its wings to get past.
Science and Technology Daily recently visited peking University's School of Life Sciences and found that researchers were trying to capture the "ideological activity" of the fruit fly, which was nailed under a microscope.
" we can directly observe changes in dopamine concentrations in the brain after fruit flies sense odors.
" Dr. Zeng Jianzhi, who is doing the experiment, said, "It will give it some electrical signal stimulation in a moment to observe the response of dopamine in the brain under different stimuli."
", the simple picture of "smell dance" is complex in the "brain map" dimension - banana sachets are captured by the olfactory cells of fruit flies, stimulating signal conversion in the olfactory cell pathway, transforming chemical signals into neuroelectrical signals to the brain, the arrival of electrical signals to urge the secretion of dopamine in the body, by opening the channels within the cell, the nerve signals in the smell of the molecule information transmission.
like a domino, the outside world's stimulation passed on, provoking the reaction of the organism.
because of the fact that instant and positioning observations of live animal thinking have not been realized for many years, and what parts of the brain these reactions occur, humans are still unknown.
, a new issue of Cell magazine published the research of the team of researchers at Peking University's School of Life Sciences, Li Weilong, who developed a dopamine fluorescent probe by genetic construction, like lighting a torch in a deep thought maze, "lighting up" the dopamine nerve loop for such related research.
dynamic tracing dopamine signal changes" combined the fluorescent genes in jellyfish with the human-sourced G protein conjugate receptor (GPCR) gene, we obtained a tool for dynamic tracing dopamine.
", Li told Science Daily, "it sounds simple, but the characteristics of neural activity put the probe under severe demands."
nerve activity is fleeting, it must react quickly; dopamine production may be trace, so it must be sharp; trace dopamine must be credible not "mistake", it must be accurate ... Only in accordance with these iron general rules, can be qualified to become a widely used research tools.
the "raw material" of the grinding tool is the GPCR that binds to dopamine.
", "It's a transmembrane protein that triggers its own conformational changes when combined with dopamine, and we think that if we can make such a change of ideas, it can be fluorescent, or it can solve the problem."
" one of the first authors of the paper, Dr. Ishii, of Peking University's School of Life Sciences, explains.
this can be colloquially compared to the dopamine "pressing" button previously only opened the door, and the new tool to be created to allow the open door to be lit at the same time.
, the team chose five GPCR subtypes as alternative raw materials. "We used molecular cloning to stitch together genes that encode fluorescent proteins with genes from five dopamine receptors, and to screen out the best subtypes by indicators such as the expression, positioning, and receptor affinity of fusion proteins,"
.
, " said Mr. Wellse. Will the gene expression after artificial "stitching"
be "recognized" by cells? The first obstacle to creating an efficient "probe" comes from the inside of the cell.
"normal GPCR is membrane protein, to detect the presence of dopamine signaloutside the cell, the protein must be on the upper membrane, after the addition of fluorescent protein, the nature of GPCR may change, can not be onthe upper membrane."
, " said Mr. Wellse.
precision-running cells after the "parts" are changed, whether the modified new protein can "inherit" the original function, whether the gene follows the coder principle transcribed into amino acid sequence after the protein assembly can complete the task of reaching the cell membrane to play a role, etc. are all in front of the task group "roadblock."
is only the first step in reaching the membrane.
"The connection of the two proteins is determined if GPCR is to accept the twisted-influence fluorescent protein that is produced after receiving the signaling molecule dopamine to reverse and glow."
", "In some cases, the receptor's conformational changes may not be effectively transmitted to fluorescent proteins to produce fluorescence changes."
", "know-one, hundred sand.
due to the grCR protein three-dimensional conformation, core group, structural force, etc. accurate understanding, Li Weilong team on its genetic level of modification and engraving, can be just right.
experiments have shown that the long-term expression of the probe for the growth of the pattern of organisms has no obvious effect.
using the probe, they detected dopamine release from brain chips in mice with electrical stimulation, and detected changes in dopamine signals associated with olfactory stimulation, visual stimulation, learning memory, and mating behavior in the brains of live fruit flies, zebrafish and mice.
the refinement, probe sensitivity is constantly "evolving" "People may never know that there is a nerve signal release here, but if there is a very sensitive probe, it may be seen."
" Wells said that after receiving the article, the team was always optimizing the probe, which is now more sensitive than in the paper.
if the human brain is likened to a contact map, what scientists want to do is follow the graph, looking for details of the corresponding neural activity such as memory, emotion, etc.
and the clarity of the future chart, whether it's snowflakes, flow-saving, symn, HD or 1080P, depends entirely on the sensitivity of the probe.
after breaking through the first barrier, the probe is "refined" optimization.
"We know how to link the fluorescent protein gene with a specific GPCR gene?" Well said, a little to the left or a little bit to the right, may also need to add a connecting bridge in the middle, the bridge and how to design, which can arrange the combination of the number of options. "We achieved the effect of probe optimization by finding sites that play a key role in the probe and dopamine response,
.
" Wells" introduction, the results of the mutation in the experiment may be good or bad, after the mutation will test its reaction with dopamine, screening out the "improved version", these "improved version" will then be combined to try, continuous screening and optimization, toward higher sensitivity "evolution".
more than 500 mutations, the team eventually obtained two versions of the probe with high, low affinity, suitable for different brain regions with dopamine release.
Li believes that probe sensitivity will likely improve in the future in the continuous optimization and screening, enabling it to trace more unknown neural pathways and discover neurological secrets that no humans have observed or know about.
capture nerve activity, from "flashing" to "colorful" because neurons during discharge burst a short peak of calcium ion concentration, neuron calcium ion imaging technology is still the most direct means of observing neural activity.
"Although calcium ion imaging can simultaneously reflect hundreds of neuronal activities, because many neurotransmitters can trigger changes in intracellular calcium signals, it is difficult to know what neurotransmitters are working through calcium imaging alone."
, " said Mr. Wellse.
has an article evaluation, with calcium imaging technology, the original silent neural activity has become a flashing spectacular image.
however, this "full flowering" imaging method is difficult to "fix" "qualitative" fine response problem.
" is the excitatory change in neurons mediated by dopamine? Does a particular neuronal cell always secrete dopamine, or do it secrete different concentrations of dopamine in stages? Is it the activity of an entire neuronal cell, or is it the activity of different synapses in neuronal cells? By answering these questions, it is possible to clarify the pathology of some neurological diseases and to develop drugs in a targeted manner.
," Li said.
if the calcium ion imaging technology solves the problem that scientists can finally "witness" neural signals in neural networks, so many pamine fluorescent probe technology is to solve the "sniper" nerve signals and the actual correspondence. At the same time
, Li's team also successfully developed for the first time a sensitive, specific, genetically coded acetylcholine fluorescent probe, and successfully detected endogenous acetylcholine signals in real-time in different biological systems, the relevant paper has been published in the journal Nature Biotechnology.
can imagine that when each neurotransmitter has a different color probe corresponding, that nerve activity will change from the previous "flash" to "colorful", different colors represent different neural activity.
have the tools at hand, people will take a big step forward in the study of getting a "brain map."
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