The overall goal of this procedure is to selectively ablate target genes during discrete phases of the condition place preference paradigm to test their contribution to this behavior across time. This is accomplished by first stereotaxic micro injecting the recombinant eno associated viral vector expressing Cree recombinase. Next, the mice are allowed to recover in their home cage for 10 to 14 days for maximal C reactivation.
The third step of the procedure is to test the mice in conditioned place preference, including the acquisition extinction and reinstatement phases. The final step of the procedure is to perform a histological examination of micro injected mice to validate correct micro injection placement. Ultimately, results can be obtained that show changes in acquisition, extinction and or reinstatement behavior in response to gene knockout through condition place preference behavioral testing.
The main advantage of this technique over existing methods like the CRE lock P mating system, is that it allows for more specific timing and anatomical specificity of the gene knockout discretely, allowing for the investigation of the loss of gene in each phase of the behavior. The implications of this technique extend towards treatment for addictive behavior because it involves better understanding the genetic basis that may underlie how exposure to contextual cues can trigger cocaine related memories that may prompt craving and relapse in addicts. To begin place a mouse cage with bedding on a heat lock to warm for postoperative recovery.
Set up an electric razor for shaving the head and ethanol and iodine to sterilize the scalp. Use 70%ethanol to wipe down ear bars and mouth hold of the stereo attacks using a five microliter Hamilton syringe. Draw up five microliters of viral vector and set it up in a syringe holder On the stereo attacks.
Place a microwaved heating pad or an electric pad on the stereo attacks after injecting the mouse with an IP injection of a ketamine xylazine cocktail. According to the text protocol, perform a tail pinch to check the level of sedation. Next, shave the head between the ears and eyes and use sterile cotton applicators to clean the area with alternating washes of ethanol and iodine.
Then transfer the mouse to the heated pad on the stereo attacks zero. The tooth bar and ear bar scales and set the mouse's teeth in the tooth bar, screw in the muzzle and the ear bars. If any wiggling or whisker movement is noted.
Administer a booster dose of ketamine xylazine cocktail. Then lubricate the eyes to prevent them from drying. Next, using a scalpel cut, open the scalp to just behind the ears.
Place bulldog clips on the corners of the scalp to peel away the skin from the skull. Making sure Bgma and Lambda are easily visible. Ensure that the suture from bgma to Lambda is aligned and reposition the head if necessary.
After confirming that the stereo attack settings are zeroed, put the needle tip in place at bma. Measure the dorsal ventral medial lateral and anterior posterior coordinates for both BMA and lambda. Then adjust the head until the ventral coordinates for BMA and lambda are within 0.02 millimeters of each other.
Recenter the needle tip on bgma, and from that point moving in the anterior posterior direction to the desired coordinate. Next drill a borehole at the desired coordinate. Checking that the needle is able to enter through uninterrupted by the skull, then lower the needle to the desired ventral coordinate.
Once the coordinate is reached, dip 0.015 millimeters below it for around 10 seconds to create a small pocket for the viral vector at a rate of roughly 0.1 microliters per minute, inject the desired volume of viral vector after waiting three minutes for the viral vector to diffuse, raise the needle tip 0.015 millimeters and wait an additional two minutes. When the injections are complete, apply bone wax to seal both holes and then suture the scalp. Finally, apply nerve block to the wounded area.
Remove the mouse from the stereo attacks and place it in a warm cage on a heat block to recover for 45 minutes to an hour. Let the mouse recover for at least 10 days to allow for maximal reactivation. Give the animal water and soften food and buprenorphine twice daily if necessary for pain after recovery, place mice into the central chamber of a three chamber place preference apparatus for a 62nd habituation period with guillotine doors closed.
After habituation, open the guillotine doors and allow the mice to freely explore all three chambers for 1200 seconds. Use Med PC four software to record the animal's time spent in each chamber using guidelines from the text protocol. Assign the mice to conditioning chambers based on their performance during the pretest.
During the next three days, inject 10 milligrams per kilogram of cocaine during a morning session and immediately combine them in their assigned chamber for 1200 seconds. Return the mice to their home cages for four hours, then inject them with saline and immediately confine the mice to the opposite chamber for 1200 seconds. To test for acquisition, place the mice in the central chamber with guillotine doors closed for a 62nd habituation period.
Open guillotine doors and allow free exploration for 1200 seconds or recording. Time spent in all chambers calculate preference by subtracting the amount of time spent in the saline pair chamber from the amount of time spent in the cocaine pair chamber. After performing CPP on micro injected mice, they're considered to have acquired preference for a particular chamber.
When cocaine preference defined as time spent in the cocaine pair chamber minus time spent in the saline pair chamber is significantly higher compared to the baseline preference score. If as a cohort control injected mice do not show a preference for the cocaine pair chamber or show, an aversion placement is verified immediately via histology, and those mice with incorrect placement are eliminated from the study. If preference is normally acquired by the control injected cohort, the paradigm is extended by beginning extinction.
Mice are considered to have extinguished the drug induced preference for a particular chamber. When cocaine preference across two consecutive extinction days is significantly lower compared to the acquisition preference score at this time, one can reinstate with a drug prime. If the preference score following administration of the drug prime is statistically indistinguishable from that at acquisition, then that cohort is said to have reinstated the drug-induced preference for the drug paired chamber using quantitative PCR.
This figure demonstrates that the gene of interest has in fact been knocked out in micro injected mice following micro injection of RAAV Cree GFP into the nucleus accumbens that have the L type calcium channel isoform, CAV 1.2 flocks of RAAV. Cree injected punches show a significant decrease in gene expression compared to mice injected with A-A-V-G-F-P shown here. Immunohistochemical analysis of the nucleus accumbens for GFP reveals proper bilateral placement of micro injected RAAV Cree Following this procedure.
Other techniques such as quantitative real-time PCR and Western bodying can be performed in order to answer questions regarding the effect of knockdown of a gene on the expression levels of other genes of interest. After Watching this video, you should have a better understanding of how to selectively ablate target genes in the mouse brain using stereotaxic microinjection viral vectors in order to selectively test the role of these genes in the acquisition extinction and reinstatement of the condition place preference paradigm.
