ИСТИНА

Modern technologies enabling precision control over the electrical activity of pecific cells or neurons in a living organism offer unique opportunities for the functional analysis of complex biological systems. Thermogenetics is a promising innovative neurostimulation technique, which enables a robust activation of cells and neurons using thermosensitive transient receptor potential (TRP) cation chan- nels. The TRP channels are three orders of magnitude more conductive than conventionally as-sociated with optogenetic channelrhodopsin (ChR) channels and can be activated by heat induced with infrared light. However, a broader application of this approach is hindered by a limited variety of suitable ion channels, as well as by low spatial and temporal resolution.In the present study, we demonstrate addressed thermogenetic activation of individual cells and neurons using infrared radiation and microwave field. We use TRPA1 channels from snakes as a thermo-genetic stimulator of Human Embryonic Kidney 293 (HEK-293) cells, mouse neurons, as well for induction of zebrafish larvae behavioral response. To investigate addressed heating of the transfected cells and neurons by infrared radiation, we used tunable radiation at wavelengths from 1.1 to 1.5 μm that the infrared radiation could be focused to a spot with the diameter about 30 μm, that guaranteed heating of single cell. The neuronal response to the activation by IR laser radiation is fully characterized by means of Ca2+ imaging technique. A unique fiberoptic probe that integrates a nitro-gen−vacancy diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with a single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing toxic effects of excessive heat. From the other hand, in specific regimes, this fiber probe can operate as the local source of the heat and microwave radiation enabling changing of the temperature and addressed activation of transfected cells and neurons. With this arsenal, we carried out a systematic, in-depth characterization of the performance of TRPA1 channels in HEK-293 cells and neurons, including accurate measurements of the pertinent Ca2+ dynamics and electrophysiological analysis of the response. Our study demonstrates that the snake TPA1 channels are ideally suited as a tool for addressed thermogenetics of individual cells, neurons in living animals. The work was supported by Russian Foundation for Basic Research (projects 14-29-07263, 14-29-07182, 16-32-80141). Research into thermogenetic stimulation of TRPA1 expressing cells has been supported by the Russian Scientific Foundation (project No. 14-14-00747).