Upconversion Nanoparticles Mediated Near Infrared-Optogenetics Manipulation Of Caenorhabditis Elegans Locomotion

Optogenetics technique utilizes light to regulate the physiological function of cells in a high temporal/spatial manner.It relies on two elements:light and light-sensitive proteins.Most of the light-sensitive proteins respond to visible light.However,the low penetration depth of visible light limits the application of optogenetics.Near infrared light?NIR?penetrates tissue deeply,yet the absence of light-sensitive proteins that respond to NIR remains a major hurdle for its application in optogenetics.Upconversion nanoparticles?UCNPs?have been applied in NIR-mediated optogenetically regulation and activation of specific neurons to induce signal transduction.However,there are limited reports on the regulation of animal motor behavior.Systematic?in-depth studies are needed to deciper the underline mechanism.In this report,we designed and synthesized a kind of UCNPs that can effectively convert 808 nm NIR light to green light emission.Such UCNPs is compatible with Chrimson,a cation channel activated by green light;as such,it can be used in the optogenetic manipulation of the motor behaviors of Caenorhabditis elegans.The main results are as follows:?1?Preparation and characterization of UCNPs.Nd³⁺sensitized upconversion nanoparticles were synthesized by a thermal decomposition method.The nanoparticles are uniform with a size of about 26 nm.They are also in good crystallinity and are?phase.They have emission at 379,408,522,541 and 655 nm,respectively,of which,541 nm is the strongest.Compared with the traditional 980 nm excitable NaYF₄:18%Yb,2%Er,the overall fluorescence intensity increases by 63%.?2?UCNPs/NIR mediated regulation of C.elegans locomotion.UCNPs+808 nm reduces the crawling rate of hpIs593 and inhibits body bending.The electrophysiological results showed that UCNPs+808 nm could activate Chrimson expressed inhibitory D-motor neuron,resulting in a decrease in the frequency of action potential release in the body wall muscles.The swimming behavior test showed that UCNP+NIR could inhibit the swing frequency of hpIs593,and the swing period was prolonged from 0.6 s to 1.14 s.For hpIs622 expressing Chrimson on excitatory intermediate neuron DVC,UCNPs+NIR can activate DVC,increase the frequency of action potential in body wall muscles and activate the background movement.?3?Uptake and metabolism of UCNPs by C.elegans.After we fed C.elegans with UCNPs for 12 hours,the fluorescence of UCNPs in the C.elegans reached saturation.After 48 hours,all the UCNPs could be excreted.The results of fluorescence co-localization and frozen section TEM showed that UCNPs were mainly distributed in the intestinal tract of the worm,and were not uptaken by intestinal cells or diffused into neurons.UCNPs uptaken in the body could still affect the movement of C.elegans.?4?The toxicity of UCNPs for C.elegans.Short-term UCNPs/NIR exposure did not damage neurons.Long-term feeding with UCNPs did not affect neuronal development.The long-term toxic effects of UCNPs were evaluated in terms of the body length,egg lay,and lifespan of C.elegans.The results showed that the worm body length and the number of eggs laid did not change.The lifespan decreased slightly with the increase of concentration,but there was no significant difference.This study shows that UCNPs/NIR system can be used to regulate neuronal activity and motor behavior of animals.It has broad application prospects in other experimental systems,and also provides new research ideas for the follow-up application of optogenetics.