Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.
The overall goal of this procedure is to study the electrical properties of excitable cells in noninvasive physiological conditions employing zebrafish embryos together with a FRET-based GEVI. This method can help answer key questions concerning the biology of excitable cells such as the neurons. For instance this methods can be used to analyze the role over the excitability in the pathogenesis of neurological disorders.
This approach employs transparent zebrafish embryos expressing FRET-based genetically-encoded voltage indicators. The approach allows the measurement of the electrical properties of the cells in vivo in a non-invasive fashion. So this method can provide insight into spinal neuron excitability in zebrafish embryos.
It can also be applied to other in-vitro or in-vivo systems amenable to imaging studies. To begin this procedure prepare the PCR mixture to amplify the Mermaid biosensor coding sequence. Heat the PCR mixture for 15 seconds at 95 degrees Celsius.
Prime it for 15 seconds at 50 degrees Celsius and elongate it for four minutes at 72 degrees Celsius for a total of 35 cycles. After that gel purify the specific blunt PCR product using a commercial gel purification kit. Then clone the DNA fragment into pCMV-SC blunt vector and linearize the Mermaid-positive plasmid with SmaI restriction enzyme for the insertion of the HuC promoter.
Set up the restriction reaction in a total volume of 20 microliters and incubate the reaction at 25 degrees Celsius for one hour. Subsequently, gel purify the digested plasmid using a commercial gel purification kit. Then PCR amplify the HuC promoter with PFU DNA polymerase and zebrafish genomic DNA as template.
Set up the PCR mixture. After an initial step of two minutes at 95 degrees Celsius, heat the PCR mixture for 15 seconds at 95 degrees Celsius. Prime it for 15 seconds at 50 degrees Celsius and elongate it for four minutes at 72 degrees Celsius for a total of 35 cycles.
Then gel purify the specific blunt PCR product. Ligate an equimolar amount of the purified pCMV-SC Mermaid and pHuC DNA using one microliter of T4 DNA ligase and one microliter of the Specific 10x buffer in a total volume of 10 microliters. Afterward incubate the reaction for 16 hours at four degrees Celsius.
Transform an aliquot of competent cells with five microliters of the ligation reaction. Select the pHuC-positive clones with the promoter inserted in the proper orientation by a CEL1-EcoRV double digestion. Transfer the clones in 15-milliliter tubes and incubate them at 37 degrees Celsius overnight.
After that extract the plasmid from the cells and set up the CEL1-EcoRV double restriction reaction and incubate it at 37 degrees Celsius for one hour. Then run the reactions onto an agarose gel. In this procedure transfer the fertilized eggs obtained from the wild type or sod1G93R adult zebrafish to a 10-millimeter Petri dish.
Rinse the embryos in cold fish water. Then immediately microinject them into the yolk with 200 picograms of pHuC-Mermaid plasmid. Next transfer the embryos to a Petri dish and incubate them in fish water at 28 degrees Celsius until they reach the desired developmental stage for the following analyses.
For a 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 jewelers forceps with sharp tips or needles. Afterward incubate the embryos for five 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 coilings 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 five micromolar riluzole. Then incubate the embryos for five 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 one percent 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 the 488 nanometer laser. Simultaneously using two photo multipliers detect the fluorescence emitted by the donor and the fluorescence emitted by the monomeric Kusibira-Orange acceptor.
At the beginning of the experiment optimize the gain and offset of photomultiplier one 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 the 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 drop-down 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. To 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 five micromolar riluzole. In this movie spontaneous coilings, motor responses that consist exclusively of a full-body contraction bringing the tip of the tail to the head were recorded at the stage of 20 to 24 hpf in regular fish medium.
This movie shows the spontaneous coilings of an embryo following the incubation in five micromolar riluzole. To test whether alterations in the electrical activity of the spinal motor neurons were at the basis of the riluzole-induced changes in the spontaneous coiling frequency, the membrane potential in embryo spinal motor neurons were studied in a completely noninvasive fashion by measuring the fluorescence intensity ratio of the donor-acceptor FRET pair of the Mermaid biosensor. Primary spinal motor neurons expressing the biosensor were identified and their the basal spontaneous depolarization activities were recorded.
Together with the reduction in the frequency of tail coiling movements, riluzole administration reduced the frequency of spontaneous depolarization events. Once mastered, this approach would allow researchers to study electrical properties of the neural networks of intact embryos which fully preserves the complexity of interactions among cells in the developing functional system. After watching this video, you should have a good understanding of how the electrical activity of the spinal neurons correlates with the frequency of the spontaneous tail coilings.
