The overall goal of this procedure is to culture dopaminergic and stri AAL Explan to study Meso Sal and Sal Nigro pathway development. This is accomplished by first micro dissecting the striatum and to dopaminergic midbrain from E 14.5 mouse embryos. The second step is to cut appropriately sized explan from the dissected tissue.
Next, the explan are positioned within a collagen gel at close proximity to one another. The final step is to culture these explan over a period of two to three days to allow for axon outgrowth. Ultimately, immunohistochemistry for specific neuronal markers is used to show and quantify attractive or repulsive axon responses.
The main advantage of this technique over existing methods like dissociated or two DX explant cultures, is that the collagen matrix provides a three dimensional substrate for the exons mimicking the extracellular environment. In addition, the collagen matrix allows for def formation of relatively stable gradients of proteins released by X explants or cells placed in the vicinity. Demonstrating the procedure will be Ava Smith and Francesca, more two grad students from my laboratory.
To begin this procedure, dissect the E 14.5 mouse embryos from the uterus of the mother. Keep them in L 15, medium on ice until needed. Next, transfer an embryo to the microscope, isolate the brain from the embryo.
Then remove the cephalon by cutting along the medial part of each cephalic vesicle. After that, remove the meningeal sheath. Make a dorso ventral cut just rostral of the meas cephalic flex.
Subsequently, make another dorsal ventral cut just coddle of the me cephalic flexor. Then make a rastro coddle cut along the dorsal midline using a microdissection knife to expose the underlying ventral midbrain tissue. Be careful not to hit the ventral midbrain tissue as it contains the dopaminergic neurons.
Afterward, make two rost coddle cuts lateral and parallel to the ventral midline to remove the dorsal midbrain tissue. Next, divide the remaining ventral midbrain tissue into X explan using a microdissection knife at the end store. The X explants in L 15 medium containing 5%FBS on ice until use.
Now transfer the talon cephalic vesicles obtained earlier during midbrain dissection to the microscope. Remove the thalamus by cutting between the thalamus and the satu using a pair of forceps. Then make a medial lateral cut rostral to the striatum to remove the rostral structures such as the olfactory bulb.
Next, repeat this step for the tissue located coddly to the striatum. After that, position the remaining slice in order to obtain a coronal view of the striatum. The striatum can be recognized as a slightly more transparent part of the tissue.
Then isolate the striatum, then cut it into implants using a microdissection knife. Avoid the slightly darker tissue close to the midline, which contains migrating neurons of the lateral and medial ganglion eminence. Add a drop of prepared collagen to a cover slip in the well of a four well no dish.
Keep it in the incubator at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. The collagen will gelatinize during this incubation. After the collagen has gelatinized, use a pipette with a 200 microliter tip to transfer the dopaminergic or sal explants to the collagen.
Then move the explants to each other in close proximity using a needle. Keep them apart at a distance of approximately the diameter of one x explan. Next, remove the excess medium.
Add 20 microliters of the prepared collagen on top of the explan. This will often cause the explants to move around. Reposition the explants using a needle.
Let the collagen solidify at room temperature for 15 minutes. Then transfer it to the 37 degree Celsius incubator supplied with 5%carbon dioxide for 30 minutes. After the collagen has set, add 400 microliters of explan medium and let it grow for two to three days in the 37 degrees Celsius incubator.
To fix the explan gently, add 4%para formaldehyde and keep them at room temperature for one hour. After one hour. Wash them in PBS at room temperature three times each time for 15 minutes.
Then after removing the PBS, incubate them in blocking buffer for two hours After that, incubate them overnight with primary antibody and blocking buffer at four degrees Celsius the following day. Wash them in PBS five times at room temperature each time for one hour. Then incubate them with secondary antibody conjugated to the appropriate flora.
Four in blocking buffer overnight at four degrees Celsius. From this step onward, cover the explants with aluminum foil in order to minimize their exposure to light. Wash them overnight in PBS at four degrees Celsius, followed by several washes the next day.
Next, mount the explants on the microscope slide using prolonged anti fade reagent mounting medium with the explant side facing down. Place a cover slip on the mounting medium very gently to avoid trapping any air under it. Then acquire digital images of the x explants using an epi fluorescence microscope.
Using these images, divide each X explan into quadrants in order to generate a proximal quadrant and a distal quadrant. After that, measure the length of the 20 longest neurites emerging from the X explant, both in the proximal and distal quadrants. Use the average length of the 20 longest neuros to calculate the proximal distal ratio for each individual explan.
Proximal distal ratio larger than one indicates axon attraction. While the ratio smaller than one indicates axon repulsion. Shown here is a midbrain explan stained with anti tyrosine hydroxylase antibody revealing axon outgrowth.
The dotted line indicates the adjacent explants. The arrows here indicate individual neurites and growth cones. Here is an example of proximal distal ratio quantification.
The are divided into equal quadrants. The proximal quadrant is facing the adjacent X explants while the distal quadrant is facing away from it. After watching this video, you should have a good understanding of how to co-culture dopaminergic and strato explants to investigate misal andal nigro pathway development, and to study the molecular mechanisms involved in information of these pathways.
