This method can help answer key questions in the genetic and neuro development such as during postnatal development. The main advantage of this technique is that it is a simple technique with home-made device. Visual demonstration of this method is critical because making targeted injections into the brain requires full attention to detail.
To begin, make a holder to use neonatal pups in a stereotaxic apparatus. First make the head tray. Cut off about 1/5 of the bottom of a 1.5 milliliter centrifuge tube to make an opening for the head of a neonatal pup.
Next, select an empty pipette tip box that fits the pedestal of the stereotaxic apparatus. Then use hot glue to secure the head tray to the base of the tip box. Next, glue a tissue-embedding cassette onto the box where it can support the pup's torso while its head is fixed.
Now prepare 30 gauge stainless steel needles for the injection. First, pre-clean them by soaking them in chloroform for three days in a glass vial. Three days later, carefully remove the needles and wash them with absolute ethanol for 20 minutes and with 50 rpm of agitation.
Then wash them three times in 70%ethanol for 10 minutes per wash also with agitation. After the washes, let the needles air dry and store them in a clean box at room temperature until use. For the microinjection assembly, use a five centimeter segment of PE20 polyethylene tubing to bridge the PE10 tubing to a 26 gauge 10 microliter syringe.
Secure the junction with cyanoacrylate. Next, mount the new 30 gauge injection needle onto the other end of the PE10 tube. Now load the 10 microliter microinjection syringe.
Remove the plunger and use another 25 gauge syringe to back load the assembly with autoclaved distilled water to remove air from the tubing. Then place the plunger back to the microliter syringe and push the plunger until only two microliters of distilled water remains in the barrel and no less. Next, carefully mount the microliter syringe onto the microflow syringe pump.
Then pipette small amounts of filtered 0.1%Fast Green dye in the experimental virus liquids onto a piece of parafilm. Now aspirate a small amount of air into the syringe until an air bubble is visible between the injection needle and the tubing. Then load 0.7 microliters of filtered Fast Green solution to test the flow of fluids in the microinjection tubing.
If the flow is good, then aspirate another small volume of air to make a second air bubble and follow by loading the injection solution into the microinjection tubing. Attach and secure the microinjection needle to the stereotaxic apparatus and proceed with the microinjection of the neonatal mice. In preparation for the surgery, thoroughly wipe down the stereotaxic instrument with 70%ethanol and sterilize the surgical instruments with 70%ethanol.
Then anesthetize a neonate using hypothermia. Place the pup in a latex glove and immerse it in crushed ice up to its neck for five minutes. Then pinch the pup's feet with forceps to make sure there is no pedal reflex.
Next, position the pup with the glove in the head tray and put some crushed ice around the latex sleeve to maintain the hypothermia anesthesia. Then scrub the pup's head with 70%ethanol and locate the lambda landmark. Mark it with a marker.
Then aim the needle tip to the lambda and set the anterior/posterior and medial/lateral coordinates to zero. Next, consulting a brain atlas, move the injection arm to the target site. For example, the striatum of P2 pups is 2.4 millimeters anterior to the lambda, 1.0 millimeters to either side of midline, and 1.7 millimeters ventral.
Next, mark the position of Fast Green dye on the PE10 tube using a pen. Then slowly begin to penetrate the needle through the skin and skull until the needle tip contacts the surface of the skull. Set the dorsal/ventral coordinate as zero.
Then lower the needle slowly to the target site. Once in position, wait for one minute to allow the parenchyma to return to its normal shape. Then run the microinjection program at 100 nanoliters per minute.
During the injection, watch the Fast Green dye move in the PE tubing. It is critical to slowly penetrate the needle through the skin and skull until the needle tip contacts the surface of the skull. During the injection, it is imperative to watch the Fast Green dye move in the PE tubing.
After the injection is completed, wait one minute and then slowly bring up the needle to 1/2 of the penetrated DV depth. Then wait another 30 seconds before proceeding with the slow withdrawal of the needle from the pup's head. After completion of the injection, it is important to wait one minute before slowly bringing up the needle to 1/2 of the penetrated DV depth.
Then wait another 30 seconds before slowly withdrawing the needle from the pup's head. After all the targeted sites have been injected, warm up the pup for 20 minutes in a 33 degree Celsius incubator. Check on the pup every five minutes until it has regained sternal recumbency.
Then return the pup to its dam. 200 nanoliters of virus that express Cre DNA recombinase fused with GFP were injected into the P2 striatum of Ai14 mice. These mice expressed the tdTomato reporter gene upon Cre-mediated deletion of a loxP flanked stop cassette.
The brains were harvested at P14 for immunostaining of GFP and tdTomato. Many AAV transduced GFP positive cells were present throughout the striatum indicating an extensive infection of striatal cells by AAV EGFP Cre viruses. Similar extensive expression of tdTomato was also found in the striatum.
Upon microscopic examination at high magnification, clearly all GFP positive striatal cells were found to co-express the tdTomato reporter gene. Moreover, tdTomato signals were detected in presumably axon terminals in the globus pallidus, in the substantia nigra pars reticulata, the target regions of striatonigral and striatopallidal projection neurons. Together, these results suggest a successful Cre loxP mediated DNA recombination induced by AAV mediated expression of Cre activity in neonatal striatal neurons.
Once mastered, this technique can be done in 30 minutes for bilateral striatal injection if it is performed properly. While attempting this procedure, it's important to remember that this is a delicate neonatal brain surgery that requires patience and carefulness. From this procedure, other methods like optogenetics and chemogenetics can be performed in order to analyze the maturation of during postnatal development.
