Establishment And Application Of Wireless Optogenetic Technology Based On Near-infrared Light Field Illumination

Optogenetics,as a neuronal regulation technique with excellent spatiotemporal specificity,has become one of the frontier technologies in the field of neuroscience and has been widely used.The wavelength of the excitation light of the Channelrhodopsins used in optogenetics is concentrated in the visible light region,and the visible light has a weak penetrating ability to biological tissues and cannot directly stimulate deep nuclei.Traditional technology usually implants a ceramic ferrule into the deep nucleus of brain tissue through surgical operation for light transmission.The implanted ceramic ferrule will cause damage to the experimental animal,and the optical fiber that transmits the stimulating light signal also limits the range of motion of the experimental animal.In order to solve the above problems,researchers have proposed a new wireless optogenetic technology based on the combination of Near-infrared(NIR)light and Upconversion nanoparticles(UCNP).This technology uses NIR light to irradiate the target brain area,and with the up-conversion ability of UCNP,the long-wavelength excitation light is up-converted into short-wavelength emission light to achieve effective stimulation of neurons in the target brain area.Since NIR light is less absorbed by biological tissues and can effectively penetrate a certain thickness of biological tissues to achieve stimulation,this technology can achieve wireless regulation of neurons in the target brain area in the study of brain functions.However,the existing UCNP-mediated wireless optogenetics technology mostly uses NIR light near the skull or NIR light combined with the automatic tracking system irradiation method,resulting in the small application range of the technology,complex experimental equipment,complex control methods,etc.adverse conditions.Therefore,in order to improve the existing wireless optogenetics technology to solve some of the problems,we propose to use the wireless optogenetics technology based on near-infrared light field irradiation,so that the wireless optogenetics technology has the characteristics of simple operation,convenient control and small damage.In this study,the experiment is divided into two parts:in vitro experiment and in vivo experiment.In order to verify the feasibility of NIR light-activated Channelrhodopsin-2(ChR2)under UCNP-mediated,we first used Transmission Electron Microscope(TEM)and Photo luminescence(PL)to characterize UCNP under in vitro conditions,and detect its emitted light Wavelength.In order to detect the biocompatibility of UCNP,UCNP is added to the normally grown cells to incubate to detect cell viability.Finally,the cells expressing ChR2 were co-incubated with UCNP for electrophysiological experiments,and the transmembrane current of the cells was recorded to study the characteristics of the transmembrane current and the intensity of the irradiation.In order to verify the feasibility of wireless optogenetics technology for NIR light field irradiation,we conducted the following experiments at the in vivo level.First,the second motor cortex(M2)of mice was injected with ChR2 viral vector and UCNP.After the virus vector expressed,the mice are grouped for experimental operations,and then the M2 brain area is subjected to immunofluorescence staining to detect the expression of c-fos,which proves that the technology can wirelessly stimulate the neurons in the target brain area under in vivo conditions and cause neurons activity.Because the M2 brain area regulates motor behavior,unilateral activation of neurons in this brain area will cause deflection movement in mice,which can easily verify the feasibility of this technology,so M2 is selected as the target brain area.Then,M2 brain area expressing light-sensitive channel protein and injected UCNP mice were lighted in a freely active state,and the total movement distance of the mice in the three stages of pretest,test and posttest was recorded.The experimental results are as follows:1.In vitro experimental resultsUCNP characterization results show that under 980nm excitation light,UCNP has obvious emission peaks at 450nm and 475nm,and its emission wavelength is consistent with the ChR2 excitation wavelength,which can be used to activate the photosensitive channel protein.Cell viability testing found that the UCNP used has good biocompatibility.UCNP has no significant effect on the viability of normal cells at concentrations of 100 ?g/ml and 20 ?g/ml,and can be used for cell and in vivo experiments.Cell electrophysiological experiments found that both 980nm NIR light mediated by UCNP and 473nm blue light can induce cells to produce transmembrane currents,and when UCNP is absent,transmembrane currents cannot be formed.The transmembrane current generated by the two kinds of light is positively correlated with the light intensity within a certain range,and a plateau(473nm:408-1170 mW,980nm:121 1-2147 mW)occurs at higher light intensity,which increases to a certain extent.The transmembrane current of cells wi th high light intensity no longer increases.Comparing the transmembrane current generated by the two wavelengths of light irradiation,it is found that there is a conspicuous difference between the two.When the transmembrane current reaches the same magnitude,the power of the 980nm NIR light source is significantly greater than the power when 473nm blue light is stimulated.Compared with the transmembrane current induced by 473nm blue light,the transmembrane current formed by cells irradiated with 980nm NIR light reaches its peak slowly,and cannot maintain a stable photocurrent.The above-mentioned in vitro experiments prove the effectiveness of the wireless optogenetic system mediated by UCNP,and at the same time suggest that our new wireless optogenetic technology is different from the traditional optogenetic technology in terms of light parameters.2.Results of in vivo experimentsThe results of immunofluorescence staining showed that both wireless optogenetics technology and traditional optogenetics technology to stimulate neurons in the M2 brain region can cause the expression of c-fos protein to prove neuronal activity.The staining results of the control group showed almost no expression or a small amount of c-fos protein,so it proved the effectiveness of wireless optogenetics technology based on near-infrared light field irradiation at the molecular level.The results of behavioral experiments showed that the total distance of the mice stimulated by the traditional optogenetic technology at 473nm increased significantly compared with the pretest and posttest,and showed obvious deflection behavior and circling movement.The total movement distance of the mice stimulated by NIR light during the test period did not increase significantly compared with the pretest and posttest(pretest:738.621 ±91.6376 cm,test:590.119±59.7591 cm,posttest:577.702±59.5587 cm),and there was no deflection Behavior,indicating that wireless optogenetics technology has failed to achieve effective regulation of mouse movement behavior.Analyzing the experimental results,it is found that the reason for the inconsistency between the results and expectations may be related to insufficient light stimulation power per unit area and light heat radiation.This study shows that wireless optogenetics technology can induce cells to produce transmembrane currents in in vitro experiments.In in vivo experiments,this technology stimulates the target brain area to cause neurons to produce activity and express c-fos protein,which proves the effectiveness of near-infrared light field regulation.Although the behavioral results failed to achieve the previous expectations and there are certain problems,the analysis shows that there is still room for improvement and perfection of the light field control mode,which can provide new research for the continued development and wide application of subsequent optogenetic technology ideas and experimental methods.