The overall goal of this procedure is to prepare whole cerebral hemisphere explan for the study of early cortical development. This is accomplished by first harvesting mouse embryos on embryonic day 13. Next, inject the plasmid DNA mixture into the cerebral ventricle and perform an ex utero electroporation.
Then prepare a whole hemisphere explan from the electroporated embryo. The final step is to fix and section the whole hemisphere explan for histological analyses. Ultimately, the consequence of mutation or toxin exposure on cortical neuron development can be evaluated by the immuno histological analyses of GFP expressing neurons.
My name is Eric Olson. I'm in the Department of Neuroscience and Physiology here at SUNY Upstate Medical University in Syracuse, and today we're gonna show you a method we developed called the whole Hemisphere X explan. The main advantage of this technique over existing methods like slice explan, is that whole hemisphere X explan are simple to prepare and provide two days of organotypic growth.
The method can help answer key questions in the neurodevelopment field, such as how a particular mutation or toxin disrupts early cortical development. Generally, individuals new to this method will struggle because preparing the whole hemisphere explan without damaging the meninges, can require some practice. Visual demonstration of this method is critical as the dissection and explan transfer steps are difficult to learn due to the sensitivity of the tissue to mechanical disruption.
Demonstrating the procedure will be Dr.Theresa Ozzi, a postdoc in my laboratory. For this protocol, use a commercially available kit to purify the plasmids. Prepare the plasmid DNA at 0.33 milligrams per milliliter in water and add fast green dye as an injection tracer at about 0.02%Weight by volume always disinfect the surgical tools, the workspace, and the microscope.
With 70%ethanol then sacrifices a required number of Swiss Webster dams on E 13 by carbon dioxide inhalation. When the embryos are removed, place them in a 10 centimeter dish with ice cold HBSS in the dish kept ice cold. Carefully dissect each embryo from the surrounding extra embryonic membranes.
Now transfer each embryo individually to a clean dish with HBSS ice cold. Using a Hamilton syringe, inject two to three microliters of the prepared DNA into the ventricle of the left cerebral hemisphere. Do not inject into or near the cortical area intended for future analysis.
To electroporated the DNA, hold the embryo gently with forceps and lightly place the positive paddle of the tweezer electrode on the dorsal midline of the head. Lightly place the negative paddle underneath the embryo chin. Run a program of five 30 volt pulses, 50 milliseconds each on one second intervals using A BTX 830 model por other systems can be set according to the manufacturer's instructions.
Return each embryo to the ice cold HBSS and proceed with the whole hemisphere Explan preparation under a dissection microscope using two number five jewelers forceps. Remove the skin and cartilaginous skull from the embryo head. Next slide of forceps underneath the brain.
To remove the intact brain from the skull, dissect away the electroporated hemisphere, including the attached midbrain tissue. Be careful not to damage the meninges overlying the dissected cortical hemisphere. We found the most difficult step to master is dissection and removal of the electro parade hemisphere without damaging the meninges.
Once dissected, transfer each hemisphere to five milliliters of ice cold HBSS using a widened one milliliter pipette tip. If needed, the embryos can be kept in separate wells of a 12 well tissue culture dish. Next, gently transfer up to six hemispheres onto a 24 millimeter collagen coated culture.
Insert and arrange the medial side down. Now fill a 35 millimeter well of a six well dish with 2.7 milliliters of supplemented media. Remove any excess HBSS from the insert and place the insert into the well with the media pipette a few drops of the media from the well onto the top of each x explan.
Do not increase the volume of media. We found that it is critical to place a few drops of culture media on top of the X explan, such that the X explants are not quite completely covered with media. This can mean adding as much as 400 microliters to the 2.7 mils of media on top of the x explan.
Move the dish into a billups rothenberg chamber that contains a humidifying dish of water and has been infused with 95%oxygen for at least a minute. Seal the chamber shut and disconnect the gas supply tube. Place the chamber into a 37 degree Celsius incubator for two days by which time prepl splitting will have taken place.
In preparation. Make pads for embedding the explan. Make a 10%calf skin gelatin solution at 60 degrees Celsius.
Once Solubilized, pour approximately 10 milliliters of the solution into a 10 centimeter dish. Keep the remaining solution at 60 degrees Celsius for later use. Allow the pads a half an hour to solidify if needed.
They can be stored at four degrees Celsius for several days, but must be warmed to room temperature before use. On the day of the fixation in a fume hood, prepare the Pagano, fixative and pagano solution without fixative. Warm the fixation solution to 37 degrees Celsius.
Retrieve the explants from the incubator without disturbing the tissues carefully and quickly. Remove most of the media and replace it with five milliliters of warmed fixative. Make sure each explant is completely submerged.
Fix the tissues for one hour at room temperature. Then remove the fixative and replace it with Pagano solution. Without fixative.
Add a few drops of 10%sodium azide to each well. The tissues can now be stored at four degrees Celsius until they're embedded on an embedding pad. Draw a grid on the bottom of the dish with the aid of a ruler.
Make two to three centimeter squares. Transfer the individual explants onto the embedding pad. Pipee away any residual solution and cover each explan with warm 10%gelatin solution.
Allow it to solidify. Once solidified slowly, add more gelatin solution until each explant is completely surrounded. Avoid melting the pad, which can occur if too much gelatin is added at once.
Now, allow for the gelatin to harden for about an hour. Once hardened, the individual explan can be cut into small blocks of gelatin before sectioning. It's important to post fix the blocks in Pagano fixative for a day or two.
When sectioning. Position the explants with the olfactory bulb up and section in the coronal plane at 100 micrometer thickness. Collect the sections in chilled PBS with 0.1%sodium azide.
Store the sections at four degrees Celsius until they can be stained using the outlined technique. Whole hemisphere explants were prepared at E 13 prior to prelate splitting. In the dorsal neocortex.
The tissue harbored a transgene that reports immature neurons of the excitatory cortical lineage. Cortical explants from the same litter were drop fixed sequentially over a period of two days in vitro and processed for subsequent histological analysis. At the initial analysis time point DIV zero prepl splitting has not yet occurred in field one by one.DIV.
The CP become apparent and it continued to grow up to 2D IV.Thus the whole hemisphere explants cultured initially at E 13, supported two days the maturation of the cerebral cortex and captured prelate splitting in the dorsal neocortex to confirm normal histological development in the dorsal neocortex. 2D IV explan were immuno stain for chondroitin sulfate, protag glycans, and extracellular matrix component. Two bands of immunoreactivity in the dorsal cortex indicated appropriate splitting of the PPP into MZ and SP during the in vitro culture period.
The PP was split by L six cortical neurons expressing CTIP two and TBR R one. The immuno staining pattern of these markers confirms the appropriate expression of these transcription factors in the newly formed cp. Together these analyses confirm ORGANOTYPIC early cortical development in the whole hemisphere explan using the entire described protocol, the left ventricle of intact E 13 embryos were injected.
Electroporated and processed GFP expression could be observed from neuronal precursors in the vz. While intense GFP expressing cells were observed in the IZ and cp, importantly numerous GFP positive neurons marked by arrowheads were observed at the top of the forming CP with emerging dendrites and axons. The discrete layer at the interface between the CP and MZ indicates appropriate migration, arrest and lamination.
The dendrites extended into the MZ while cortico ugal axons extended below the CP and into the iz.Below. The differentiating cells were GFP positive cells in varying states of maturity, including neurons with a bipolar morphology juxtaposed to radio glial fibers and below them multipolar neurons in the iz. The dendrites also extended into the MZ where the ECM protein readin is appropriately immuno localized.
Thus, the electroporation and explant conditions permit for normal development of cortical neurons. As expected post mitotic GFP positive neurons populated the forming cp. Thus, the protocol can reliably target L six cortical neurons for studies of their migration and maturation.
Extensive use of this procedure has resulted in a 79%success rate. 195 of 248 attempts have been suitable for imaging and analysis. Half of the failures came from a failure to express GFP or missed targeted GFP Once mastered.
This technique can be done in 90 minutes if performed properly. While attempting this procedure, it is important to remember to practice the dissections so that the electroporated hemispheres can be cultured with intact meninges Following this procedure. Other methods like time lapse microscopy can be performed in order to answer additional questions like how mutations of interest disrupt neuronal migration or dendri agenesis.
