Optogenetic regulation of leg movement in midstage chick embryos through peripheral nerve stimulation

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