A Non-invasive Approach to Study the Electrophysiology of Motor Neurons in Zebrafish Embryos

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.

For spontaneous tail-coiling analysis, transfer the embryos to a 90-millimeter round Petri dish filled with fish water containing 0.2% DMSO, and manually dechorionate them using two jeweler's forceps with sharp tips or needles. Afterward, incubate the embryos for 5 minutes at 28 degrees Celsius. Using a digital camera mounted on a stereomicroscope, detect the tail coiling at room temperature during a one-minute video recording.

Next, acquire the time series at a time resolution of 30 frames per second. Then, calculate the frequency of spontaneous tail coiling by counting the number of bends per time unit. To evaluate the effect of the drug riluzole, transfer the embryos to a new 90-millimeter Petri dish filled with fish water containing 5 micromolar riluzole. Then, incubate the embryos for 5 minutes at 28 degrees Celsius before recording a one-minute video and performing the behavioral analysis.

Now, mount the 20 to 24 hpf embryos in 1% low melting point agarose in fish water inside a 35-millimeter glass-bottomed imaging dish at 37 degrees Celsius. Orient the embryos on their sides and wait until the agarose solidifies at room temperature.

Next, transfer the imaging dish to the stage of a confocal microscope. Identify the motor neurons expressing the biosensor by exciting monomeric Umikinoko-Green with 488-nanometer argon laser. Then, set its emission between 495 and 525 nanometers and press Record.

For a FRET measurement, excite mUKG, the donor of the FRET pair with a 488-nanometer laser. Simultaneously, using two photomultipliers, detect the fluorescence emitted by the donor and the fluorescence emitted by the monomeric Kusabira-Orange acceptor. At the beginning of the experiment, optimize the gain and offset of photomultiplier 1, where the mUKG fluorescence is recorded, and keep them constant throughout the session.

To set the offset, change the color of the image to the intensity values by using the Q-lookup table. While scanning with a laser off, use the offset slider so that the background pixels have an intensity slightly higher than zero. Using the same lookup table, switch the laser on. While scanning, use the gain slider to maximize the signal-to-noise ratio, being careful to avoid saturated pixels.

In the software acquisition window, select the xyt acquisition mode and an image field size of 512-by-64 pixels from the dropdown menu. Then, set the recording of the changes in embryo spinal neuron voltage to acquire a single xy plane every 30 milliseconds for one minute. Evaluate the effect of riluzole administration on membrane depolarization in the same neuron, acquire a new data set five minutes after the addition of fish water containing 5 micromolar riluzole.