Thermogenetics instead of optogenetics
The neural network can be controlled using infrared radiation
Employees of the M. M. Shemyakin and Yu. A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, together with colleagues from the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences and Moscow State University, have developed a method for stimulating nerve cells with infrared radiation.
To do this, they embedded snake proteins that react to temperature – thermoreceptors - into the neurons of mice (in cell culture) and live danio rerio fry. The results published in the journal Nature Communications (Ermakova et al., Thermogenetic neurostimulation with single-cell resolution) will help stimulate neural networks in deep tissue layers without implantation into the body, as well as control the activity of other cell types in living systems. In the future, this may help in the treatment of various diseases of the nervous system. The results of the work are briefly reported in the press release of the IBH RAS.
"Scientists have long been interested in the question of how to "point" control neurons," says Yulia Ermakova, an employee of the Laboratory of Molecular Technologies of the IBH RAS, the first author of the article. – In 1979, Francis Crick, one of the discoverers of the structure of DNA, suggested that the main challenge in neuroscience is to create methods that would stimulate a certain type of nerve cells, while other cells would remain insensitive to the stimulus. Electrodes and medications could not cope with this – the tools were too rough and inaccurate. Crick believed that light would be suitable for these purposes. The scientist's idea was realized only in 2005, when a group of researchers from Stanford University, led by Karl Disseroth, was able to change neurons by genetically engineering methods and excite nerve cells by irradiating them with light. This method was called optogenetics – a combination of optics and genetics."
Neurons acquire sensitivity to light due to artificially placed receptor proteins in them, in nature they help living organisms navigate in the environment. Depending on the types of physical effects, the receptors are divided into different classes. Thus, light signals are perceived by rhodopsins and phototropins, and temperature fluctuations are perceived by thermoreceptors of the TRP (Transient receptor potential) family. It is with their help that we feel hot or cold objects, as well as the taste of spicy food or menthol "chill". Thermoreceptors formed the basis of the thermogenetics method, which also allows to "point" neurons with long-wave infrared radiation, much deeper than visible light penetrating into tissues.
The authors of the current study used TRPA1 snake thermoreceptors as receptor proteins, which are responsible for thermal vision - the ability of some snakes to "see" warm objects at a distance. This helps animals navigate in space and hunt in the dark.
The first part of the experiments was carried out on the culture of mouse neuron cells. Infrared light was applied to neurons from a fiber-optic laser installation. Their activation was recorded by measuring the flow of ions passing through the cell envelope – the main participants in signal transmission in the cell.
"So we found that the activation of the thermosensitive TRPA1 channels occurs within the first milliseconds after the laser pulse is applied," explains Yulia. "This makes it possible to use thermogenetics to rapidly stimulate neurons and reproduce complex combinations of different pulses at a speed of up to 50 pulses per second."
To confirm that thermogenetics can be used to stimulate behavioral reactions in a living organism (in vivo), the researchers conducted an experiment on Danio rerio fish, which were divided into two groups. Fish from the experimental group had snake thermoreceptors TRPA1 embedded in certain neurons in the cell shell, and from the control group – only fluorescent ("glowing") the label. After that, the researchers raised the body temperature of the fish at a certain point using a beam of infrared light (60 micrometers in diameter, which is slightly larger than the size of a large neuron). In response to this, the experimental fish experienced a false sense of touch and tried to swim away, making a reflex swing with their tail, while the second group of fish was completely insensitive to the effects of the laser.
The study included not only a biological, but also a physical component. For thermogenetic stimulation, it is extremely important to heat the living tissue at a set temperature not exceeding 1-2 degrees. Insufficient heating is not able to activate neurons. Excessive will lead to overheating and death of neurons. Therefore, a team from Moscow State University led by Alexey Zheltikov developed a method for local temperature detection using quantum effects in diamond microparticles that had special crystal lattice defects. Such a diamond placed on the tip of an optical fiber is able to measure the temperature of the heated sample with high accuracy.
"The thermogenetics method opens up broad prospects for its use in science and for further development," says Vsevolod Belousov, head of the research, Head of the Laboratory of Molecular Technologies of the IBH RAS. – Firstly, IR radiation penetrates deeper into the tissue, which means that it will be possible to stimulate deeper layers of the brain. Moreover, not only infrared radiation can be used for heating, but also focused microwave waves or high-power magnets. Secondly, thermogenetics has a huge advantage in working with small model animals, such as fish fry or fruit flies. In classical optogenetic experiments, they see blue light used to activate neurons and are frightened by it. IR radiation is invisible to them, so you can not be afraid of adverse reactions of the animal to bright light. Thirdly, the obtained molecular and technical tools can be used to activate not only neurons, but also other cells. All together, this will lead to the emergence of new approaches to therapeutic stimulation or, conversely, suppression of the functions of various cells in the body."
Optogenetics is taking its first steps in this field, and now thermogenetics, which has been brought to a new level by this work, will join it.
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23.05.2017