Optogenetic Regulation of Leg Movement in Mid-stage Chick

22 Numerous disorders that affect proper development, including the structure and 23 function of the nervous system, are associated with altered embryonic movement. 24 Ongoing challenges are to understand in detail how embryonic movement is generated 25 and to better understand the linkage between proper movement and normal nervous 26 system function. Controlled manipulation of embryonic limb movement and neuronal 27 activity in order to assess shortand long-term outcomes can be difficult. Optogenetics 28 is a powerful new approach to modulate neuronal activity in vivo. In this study, we have 29 used an optogenetics approach to activate peripheral motor axons and thus alter leg 30 motility in the embryonic chick. We used electroporation of a transposon-based 31 expression system to produce ChIEF, a channelrhodopsin-2 variant, into the 32 lumbosacral spinal cord of chick embryos. The transposon-based system allows for 33 stable incorporation of transgenes into the genomic DNA of recipient cells. ChIEF 34 protein is detectable within 24 hours of electroporation, is largely membrane-localized, 35 and is found throughout embryonic development in both central and peripheral 36 processes. The optical clarity of thin embryonic tissue allows detailed innervation 37 patterns of ChIEF-containing motor axons to be visualized in the living embryo in ovo 38 and pulses of blue light delivered to the thigh can elicit stereotyped flexures of the leg 39 when the embryo is at rest. Continuous illumination can disrupt full extension of the leg 40 during spontaneous movements. Therefore, our results establish an optogenetics 41 approach to alter normal peripheral axon function and to probe the role of movement 42 and neuronal activity in sensorimotor development throughout embryogenesis. 43