The overall goal of this procedure is to visualize and analyze the migration of Neuroblast along the rostral migratory stream in the postnatal mouse brain. This is accomplished by first Electroporated, A DNA plasmid ENC coating green fluorescent protein in the lateral ventricle of the mouse pops. The second step is to prepare brain slices from the electroporated mice.
Next, the slices with the best GFP signal are selected for culturing. The final step is time-lapse imaging of the brain slices containing the GFP labeled migratory neuroblast using a spinning disc confocal microscope. Ultimately, the time-lapse movies are used for cell tracking and quantitative analysis of neuroblast migration.
This method can help answer key questions in the neurogenesis field, such as how the movement of stem cell derived neuroblast is guided and regulated in the postnatal brain. The main advantage of this technique over existing methods like viral vector delivery is that it is easier, cheaper, and less time consuming. In addition, it allows sparse labeling of neuroblast, therefore enabling a detailed analysis of their migration dynamics.
Begin this procedure by setting the voltage of the electroporated to five square pulses, 50 milliseconds per pulse at 100 volts with 850 millisecond intervals. Then load a capillary needle with one to two microliters of DNA using an aspirator tube connected to the capillary. Next, remove a postnatal day two mouse pup from the cage and anesthetize it by isof fluorine inhalation.
After that, hold the head of the pup between your thumb and index finger of your less dominant hand. Slightly pull the skin back on the head to help the identification of the right injection point. Consider a virtual line between the eye and the cranio metric landmark Lambda.
Then insert the capillary needle at about one third of the length of this line from the Lambda to about two millimeters deep afterward, inject plasmid by blowing slowly through the mouth. During this procedure, make sure your fingers are not applying too much pressure on the brain. Stop the injection when a minimal amount of DNA solution is left in the capillary.
Then coat both electrodes with gel and place them with the positive side on the lateral side of the hemisphere where the DNA was injected. For DNA incorporation into the Rostral SVZ place the electrodes slightly rostral to the injection point. Next, initiate current transfer by pressing the pulse foot switch pedal.
Then wipe off the gel from the pup's head, reanimate the pup with oxygen on a heating pad for a few minutes. Subsequently, return it to the cage and place it away from the mother. Make sure the mother retrieves the pup and reunites it with the rest of the litter.
In this procedure place milli cell inserts into a 35 millimeter glass bottom culture dish containing one milliliter of movie medium. Then place the culture dish in a humidified incubator at 37 degrees Celsius, 5%carbon dioxide. After decapitating the mouse pup, remove the scalp with a scalpel.
Next, cut the skull along the mid sagittal suture from the cerebellum to the olfactory bulb. Make some additional transversal cuts on the skull and gently remove the cranial flaps using forceps. Make sure that the entire brain is exposed and carefully dissect it out using a spatula, taking special care not to damage the tissue.
After that hemis dissect the brain with a razor blade and discard the unin injected hemisphere. Subsequently placed a small piece of tape on the vibram holder. Use the minimal amount of glue necessary to attach the brain hemisphere on it.
Begin cutting the brain hemisphere to 300 micron slices. Then collect the slices using a small paint paintbrush or a soft inoculating loop. Afterward, check both sides of the slices under a standard fluorescent microscope for GFP signal and choose the ones showing bright fluorescence along most of the rostral migratory stream.
In a self culture hood, cut away the most coddle third of the brain slice and remove any glue traces. Next, aspirate the slice using a plastic past pipette and place it on the center of a prewarm milli cell insert. Remove excess dissection solution on top of the insert with a gilson pipette.
Then leave the slice cultures to settle in a 37 degree Celsius 5%carbon dioxide incubator for at least one hour. Before imaging within two hours of brain slice preparation. Place the glass bottom dish containing the brain slice in the imaging chamber on the microscope stage.
Next, use a 20 x 0.45 objective under the appropriate fluorescent light to select and focus the area of the slice that will be imaged. To set up the time-lapse imaging on the microscope, click on the L 100 button to allow laser scanning of the sample. Next in velocity, open the ultra view laser changer by selecting the appropriate laser.
Set the exposure time gain and the laser intensity. Then select the Zack interval to image inside the brain. Afterward, select the spacing between each ZS stack image.
Choose the time interval between each ZS stack capture and the total imaging duration. Then click on the save icon to save the changes to the imaging parameters. At the end, press the recording button to start imaging in the Velocity quantitation module.
Open the desired library created. After completing a time-lapse experiment, select extended focus from the top left box. Click on the measurements tab to display the measurement window.
A list of tasks is visible at the bottom left of the screen. Drag track on the space above. This will prompt the opening of a new window called track.
At the top left section of the screen, select points from the input tab in the track window. Click on the point tool, click on the tools tab and select the track objects manually option a window will pop up to allow the operator to start tracking the cells. Start tracking the migrating neuroblast by clicking with the mouse on the central area of the cell body and keep tracking the cell movement until the last time point of the time lapse is reached.
To obtain data, choose make measurement item. From the measurements menu, a window will appear at the center of the screen. In this window, select a new measurement item called and type a name a measurement item.
File containing parameters for quantitative analysis will appear under the time-lapse file. Double click on this measurement item file to open it as a window to visualize the single tracks of analyzed cells. Choose tracked point from the display options and select the cells that need to be visualized for their migratory path.
To analyze migration parameters right click on the measurement item file and export it as a text file, which can then be imported in programs like Excel. These are the spinning disc time-lapse images of neuroblast taken from a mouse sagittal brain slice. Five days after electroporation of A GFP expressing plasmid images are one hour apart.
Each panel is a Zack projection of 28 consecutive images four microns apart. The arrowheads indicate four representative neuroblast migrating along the RMS towards the olfactory bulb. These are the representative migratory paths obtained from the time-lapse imaging of four neuroblast.
This graph shows the distance migrated with time of two representative neuroblast. These cells display a typical saltatory motile behavior. This movie shows a section of the mouse RMS with GFP labeled migrating.
Neuroblast obtained five days after electroporation of A GFP expressing plasmid. The olfactory bulb is located out of view towards the bottom right corner. The confocal Z stacks were captured on a spinning disc confocal.
With a 20 x objective every three minutes for three hours over a 120 micron interval, the playing speed is 10 frames per second. While attempting this procedure, it's important to remember to practice electroporation before starting experiments and also to carefully prepare the acute brain slice cultures in the shortest possible time. After watching this video, you should have a good understanding of how to visualize and analyze the Neuroblast migration in the postnatal mouse brain.
