A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos
Transcription
Place genetically engineered zebrafish embryos under a stereomicroscope and observe the spontaneous tail coiling triggered by spinal cord motor neurons.
The intrinsic properties of these neurons cause ion influx through sodium channels, leading to spontaneous membrane depolarization and the generation of action potentials that propagate to muscles, triggering tail coiling. Measure the coiling frequency.
Transfer some embryos to a sodium channel blocker to inhibit sodium influx and assess the reduced coiling frequency.
Mount untreated embryos in molten agarose; let agarose solidify to restrict movement, and observe under a fluorescence microscope.
The motor neurons express a biosensor with a membrane-bound voltage-sensing domain, or VSD, fused to donor and acceptor fluorophores.
Depolarization changes the VSD conformation, bringing the fluorophores closer.
Upon excitation, the donor transfers energy to the acceptor, causing acceptor fluorescence emission.
Add the blocker to inhibit depolarization, which reduces acceptor emission, confirming blocker-mediated inhibition of spontaneous depolarization in the neurons.
