This protocol provides a rapid way for evaluating the effects of the knock down of the WC5 gene, which is the most common cause of focal epilepsy. The main advantage of this technique is that we can use it to assess the epilepsy, like phenotype using both behavioral and physiological features at various stages of development. One day before the micro injection, set up the Zebrafish mating tanks.
On the morning of the injection, remove the dividers to enable spawning. Use a fine sieve to transfer the eggs into 100 millimeter Petri dishes, filled with embryo water. Using a plastic Pasteur pipet, Pick 60 to 80 eggs, and arrange the eggs in a silicone coated Petri dish for the injection.
we move most of the water, leaving just enough to cover the eggs halfway. Place a glass needle vertically into a tube of injection solution. Allow the colored solution to fill the tube by capillary action over a period of several minutes.
When the injection solution is visible in the tip of the needle, mount the needle onto the injecting handle of the micro injector, and turn on the air compressor. Adjust the pressure setting to generate a two nanoliter injection volume. And to place the eggs under a Dissecting Binocular Microscope, with a four times magnification.
Insert the needle tip through the chorion and the yolk of single stage embryos to inject the solution directly within each cell. Then transfer the injected embryos into a labeled 100 millimeter Petri dish of embryo water, and place the plate in a 28 degrees Celsius incubator. 28 hours post fertilization, place a plastic 1.2 by 1.2 millimeter mesh grid at the bottom of the test dish.
Use a plastic Pasteur pipette to place 10 to 12 embryos within their chorions onto the plastic mesh. Fill the dish with enough embryo water to keep the embryo submerged, but not floating, carefully moving the embryos with a plastic tip into position on the grid as necessary. Then, using a video camera attached to a Dissection Microscope, record the spontaneous coiling activity for 10 to 20 minutes.
To analyze the total spontaneous movement, use the activity quantification module in a Zebra lab system to upload the recorded video and to design the tracking arenas around each embryo as appropriate. Set the freeze and burst thresholds to 10 and 50 respectively. And when the automated video analysis, which quantifies the total activity inside each of the defined arenas.
Then recover the data set as a spreadsheet to perform the analysis, using the appropriate data analysis software. 46 hours post fertilization, use fine forceps to dechorionate the embryos, and fill a 130 millimeter test dish with embryo water. At least 15 minutes before the rest, warm the test dish in a 28 degrees Celsius incubator.
To perform a touch evoked escape response test, use a plastic Pasteur pipette to place an embryo in the center of the test dish, under the camera. Begin the recording, using an acquisition rate of 30 frames per second. Using a fine plastic tip, slightly touch the tail of the embryo with a flicking motion.
Stopping the recording, when the larva has terminated its movement. Then, transfer the embryo to a new holding dish filled with fresh embryo water, and repeat the test with as many embryos as needed for each experimental condition. To prepare the Zebra fish for electrophysiological analysis, place one, four to six days post fertilization fish in a glass bottom Petri dish.
Remove any excess, extra sailor medium to ensure that the fish will be as close to the bottom of the dish as possible. Use a Plastic pasteur pipette to add enough warm liquid agros to cover the larva. While the agros hardnes, use fine forceps to orient the fish ventral side down in the center of the dish.
Then add two milliliters of recording solution, containing 10 micromolar pancuronium bromide to the dish to block neuromuscular transmission. Next, fill a micropipetting with recording solution, and use a patch clamp amplifier in the voltage clamp configuration, to measure the electrode resistance in the bath to confirm its correct value. Using a 20x objective, position the head of the larva in the central field of view, and lower the micropipet to reach the recording position within the optic tectum.
switch the patch clamp amplifier to the current clamp configuration and fix the holding current to zero milliamp. Using a low pass filter of one kilohertz, and acquisition rate of one kilohertz, and a digital gain of 10, record the spontaneous activity of the fish for 60 minutes to determine the baseline activity levels. At the end of the baseline recording, at 143 microliters of a 300 millimolar per liter pentylenetetrazole solution to the bath, for a final concentration of 20 millimolar per liter.
Record the neuronal activity of the animal in pentylenetetrazole solution for another 120 minutes. During the baseline period of the recording, four to six days post fertilization epileptic models zebra fish demonstrate a higher occurrence of spontaneous events, while mismatch control fish display very few fluctuations. After pentylenetetrazole application, both mismatch control and epileptic models zebra fish exhibit an increased number of deep polarization events.
During the first period after pentylenetetrazole application, a rate of 0.8 events per minute is observed in both mismatch control and knock down animals for which the majority of events are of a high amplitude. During the latter response period. The rate of depolarization events increases to around one event per minute.
And the majority of the events are of a low amplitude. When performing knock down, it is very important to establish the correct dose of morpholinos using a dose response curve and to perform controls to avoid the nonspecific toxicities. This push to do enable several downstream studies, including testing the effects of genetic or chemical modifiers.
And the Zebrafish behaviour and neural activities activity to understand the developmental effects of the DC five mutations.
