The overall goal of this procedure is to label specific subsets of neurons in the chick hind brain and to trace their axonal trajectories and targets. This is accomplished by first opening a small oval window at the center of an embryonic day.2.75. Egg next DNA plasmids are injected into the hind brainin lumen.
Then the hind brain is unilaterally electroporated to allow expression of the constructs in the desired neuronal group and the eggs incubated. Finally, the brainstem is dissected from the whole embryo for flat mount preparation or tissue sectioning. Ultimately, results can be obtained that show axonal projections and synaptic targets in the hind, brainin and cerebellum at various developmental stages through immunofluorescent staining and microscopy analysis.
This method can help answer key questions in the field of developmental neurobiology, such as how specific neuronal cells project their axons and form synaptic connections during embryonic development. To prepare for electroporation, incubate them at 37 to 38 and a half degrees Celsius for 65 to 70 hours until they reach stage 16 to 17. While the eggs are incubating, pull glass capillaries connect bent L-shaped gold electrodes with an adapter holder to the pulse narrator.
Set the electroporation parameters to 25 volts, five pulse numbers, 45 millisecond pulse length and 300 pulse intervals. Prepare a mouth pipette a 10 milliliter syringe needle paraform strips a soft brush, sharp dissecting scissors, and 25 milliliters of sterile PBS. Prepare an adequate mixture of DNA plasmids and add fast green dye to visualize the injected DNA.
When the eggs have reached the proper developmental stage, remove them from the incubator and keep them in horizontal position. Use 70%ethanol to clean the eggshells. Handle one egg at a time to minimize embryonic exposure.
Time to air with the tips of the scissors. Make a hole at the egg pole and with the syringe, remove three to four milliliters of albumin. Using the dissecting scissors, open a small oval window at the upper horizontal pole of the eggshell.
During the entire process drip a few drops of PBS on top of the embryo to prevent dryness. Using a mouth pipette load a small amount of the DNA mixture into the glass capillary. Next, place the embryo with its posterior end indirect view.
Then holding the capillary at about 45 degrees, penetrate the coddle hind brain. Do not remove any membrane from around the embryo without forming any air bubbles. Carefully exhale to inject the DNA solution into the hind brainin lumen.
The solution will spread anteriorly immediately after the DNA injection. Place the parallel electrodes at the precise targeted AP and DV hind brainin positions. Without touching the heart, push the electrodes slightly ventrally and pulse the electro.
The electrodes should be covered with bubbles to indicate electrical conductivity, carefully remove the electrodes and drip a few drops of PBS to cool the embryo and to remove bubbles. Use a perfil strip to seal the egg opening properly to prevent drying during incubation with a soft brush and PBS wash the electrodes, place the egg in the incubator for the desired length of time. Survival of embryos is approximately 90%two to three days after electroporation and reduces up to 50%12 days following electroporation.
To make flat mount preparations of the hind brain, carefully dissect the embryo, separating it from surrounding membranes and blood vessels. Place the embryo in a small silicone coated Petri dish containing PBS with the embryo facing dorsally. Use tungsten pins to attach it to the dish.
Then using a sharp glass capillary. Make a dorsal slit in the hind brainin roof plate with sharp tweezers and micro scissors. Hold the upper part of the head and pull the hind brain tissue very carefully from rostral to toddle.
After separation of the hind brain, remove remaining ventral tissues. Incubate the hind brain in 4%paraform aldehyde or PFA solution for one to two hours at room temperature. Then wash it with PBT to remove the PFA.
After following the immunofluorescence preparation described in the text protocol, place the hind brain facing dorsally on a glass slide. Add fluorescent mounting medium onto the hind brain and use glass capillaries or tungsten needles to flatten the hind brain on the slide. Add a small amount of grease at each corner to prevent squeezing of the tissue by the cover slip.
Carefully place a cover slip on top of the hind brain avoiding air bubbles for adherence. Add nail polish to the cover glass edges. Cryo sections of the brainstem can be performed at any developmental stage yet after E 6.5.
They should be the preferred method due to the difficulty in visualizing axons in the flat mounted embryo, which becomes very thick. Moreover, demonstration of synapsis requires sectioning of the tissue and the high magnification view under a microscope. To prepare a sample for cryo section, dissect the embryo and remove all tissues surrounding the brain.
Rinse with PBS. Then use micro scissors and sharp tweezers to cut the embryo at the coddle region of the medulla. Make a wide incision between the cerebellum and the midbrain and remove the entire brainstem.
The tissue should contain the medulla pons and cerebellum em. In the following example, an enhancer element that is expressed in the DA one subgroup of inter neurons in the check hind brain was cloned upstream of Cree recombinase and co electroporated with a Cree dependent cytoplasmic GFP reporter plasmid. A flat mount preparation at stage E 4.5 reveals two contralateral ascending axonal projection patterns to map the axonal projections of DA one inter neurons at later stages of development.
A pre-con unconditional GFP reporter cassette was cloned between two piggyback arms, along with the A TOH one creen enhancer and transpose vector. Using this strategy, the Electroporated C precon conditional MG FP is integrated into the chick chromosomes and consequently, the stop cassette is removed only in D one neurons enabling prolonged expression of GFP. In DA one cells.
The mesylated form of the GFP tethers it to the membrane such that it is localized along the axonal membrane rather than being diluted in the cytoplasm. In braint stems collected at E 13.5 and analyzed in sagittal sections. DA one axons were found to accumulate in the cerebellum and extend towards the external granular layer or EGL, which is marked by ZI one positive cells.
Synaptic targets of D one axons in the cerebellum were also analyzed. E 2.75 embryos were electroporated with synaptic vesicle protein two reporter plasmid, along with a H one Cree enhancer and the piggyback transposes. This method enables the expression of the GFP reporter in the presynaptic DA one axonal termini.
The cerebellum was sectioned at E 13.5 and stained with the general presynaptic marker synaptic taman, as well as with calbindin that labels the bikini layer. A high magnification view of the section demonstrates SV two GFP positive synaptic vesicles in multiple cerebellar regions, including the bikini layer. This result reveals the synaptic connections of DA one hind brainin inter neurons in the cerebellum.
After watching this video, you should have a good understanding of how to electro operate DNA into the avian Hein at embryonic day two point 75 in order to specifically label a subset of neuronal cells. This procedure enables one to follow the axonal projections and synaptic connections of the labeled neuronal cells at multiple developmental stages.
