The overall goal of this procedure is to assess how modulating the expression of a gene in a target area affects animal behavior. This is accomplished by first creating a lentivirus to modulate the expression of the target gene. The second step is to use stereotactic injection to inject the lentivirus into a specific brain area of ER rodent.
Next behaviors such as anxiety and depression, can be quantitatively measured using established behavioral paradigms. The final step is to conduct histology to confirm the correct injection site. Ultimately, stereotactic injection paired with behavioral assessment can show how a specific gene in a target area can affect mouse behaviors.
The main advantage of this technique over existing methods is that scientists can change the expression of a single gene in a small targeted area, and then evaluate behavioral changes in mice. To begin this procedure, place an anesthetized mouse in the stereotactic apparatus, latch the front teeth onto the anterior clamp and secure the jaw. Then insert the ear bars into the ear canals in order to fully stabilize the head.
Next place a heating pad under the mouse to regulate its body temperature. Throughout the procedure, place artificial tears on the eyes of the rodent to prevent drying. After that, clean the surgical area thoroughly by first rubbing 10%povidone iodine with a cotton swab in circular motions.
Subsequently rub 70%ethyl alcohol on the surgical site in the same fashion. Repeat this cleaning procedure two more times. Once the mouse is prepped, open the sterile equipment bag.
Change the gloves before touching the instruments. Then take out the sterile cloth and place the instruments on the cloth. Now gently grip the skin of the mouse with the blood end forceps and make it incision using a scalpel.
Begin the incision about 1.5 centimeters above the ears and extend to about 0.5 centimeters below the ears to expose the bgma. Once the bgma is visualized, use a sterile cotton tip to gently remove any blood covering the surface of the skull. Then use two cotton tips to push the skin toward the sides of the head.
Next, in the fume hood, fill a syringe with the virus to be injected. Ensure that no bubbles are trapped inside. Then place a syringe in the stereotactic apparatus, making sure that it is fully secured.
After that, slowly lower the syringe until it is right above the surface of the skull. Then move the tip of the sterile syringe needle to the intersection of B bgma and the inter rural line. Set this point as zero.
Subsequently set the coordinates of the area of interest. Move the needle of the syringe to the target site to be drilled. Then place the drill bit right above the target site at a 45 degree angle to the skull and begin drilling.
Be careful not to drill into the brain in order to prevent cortical damage. After that, clean away any blood from the hole with a sterile cotton tip. Lower the syringe so that the tip sits right on the surface of the brain.
Set the DV coordinate to zero, then lower the syringe to the desired depth. Now start the injection at a rate of 0.2 microliters per minute. After the injection is finished, wait about two minutes to ensure that any residual virus has been absorbed.
Then slowly raise a syringe. Use a cotton tip to clean away any fluid from the injection site. Afterward, suture the skin and remove the animal from the stereotactic system.
Keep the mouse on the heating pad until it wakes up and administer additional analgesic when needed. In this procedure, obtain a plastic square arena. Clean the arena with 70%ethyl alcohol before the experiment.
Then place the arena on the floor and ensure that the lighting is set to minimize shadows and glare. Next, adjust the detection settings so that the mouse is visible on the screen. After that, turn on the camera to record the behavioral task and place the mouse toward the middle front of the arena so that the mouse is facing the wall.
Set the timer for five minutes and step away from the arena or leave the room. After five minutes, transfer the mouse back to the cage. Use 70%ethyl alcohol to thoroughly clean the field before proceeding to the next animal.
Now fill a two liter clear bucket with water at 22 degrees. Place the bucket on the floor and ensure that the lighting is set to minimize the shadows and glare. If using tracking software, position the camera directly above the bucket.
Adjust the software settings so that the mouse is easily visible on the screen. Then turn on the camera to record the behavioral task. Gently place the mouse in the middle of the bucket of water so that its front feet.
Touch the water first and prevent its head from submerging. Afterward, set the timer for six minutes before stepping away from the behavioral area. After six minutes, remove the mouse from the bucket, wipe off excess water before placing it back in the cage.
Here is a representative brain slice, which illustrates the successful injection of virus into the dentate gyrus. This figure shows the amount of time that the control and experimental mice spent in the center of a novel open field. The experimental mice spent significantly less time in the center of the arena indicating a phenotype of anxiety, and this figure shows the amount of time that the control and experimental mice are immobile in the forced swim test.
The experimental mice were not significantly different from the controls in the amount of time spent immobile. After watching this video, viewers should have a good idea of how to use stereotactic injections to assess rodent behaviors.
