HSV-Mediated Transgene Expressie van Chimere constructen tot Behavioral functie van GPCR heteromeren in Muizen Studie

The overall goal of this method is to observe how virus-mediated gene transfer affects behavioral phenotypes that require heterodimerization of G-protein coupled receptors in mouse models. This method can help answer key questions in the field of neuro and psycho pharmacology. Specifically how g protein coupled receptors interact with each other at a molecular level.

By using this method we can understand how this interaction affects behavior in models of psychiatric disorders, such as schizophrenia and depression. Using this method we are going to specifically look at seratonin 2a and metabotropic glutamate receptor 2, both of which have been shown to be implicated in the mechanism of action of drugs used to treat patients with schizophrenia. Begin by properly anesthetizing the mouse according to approved methods.

Ensure that a surgical plane of anesthesia has been obtained by performing a toe pinch and noting the absence of a response. Then apply ophthalmic gel to protect the eyes. Attach the mouse to the stereotactic frame, making sure to adjust the tilt of the head so that the skull is level and flat.

Apply povidone iodine to the exposed scalp. Using a scalpel make a sagittal incision along the midline of the skull within the exposed shaved area. Then attach pureit clamps to the skin at the incision site to make sure that the skull remains exposed.

Apply hydrogen peroxide to dissolve away the periosteum, and expose the sutures of the skull. Now that the bregma and sutures are visible, be sure to adjust the stereotactic frame to make sure that the skull is level. Align the needle tips of the syringes with bregma and record the coordinates of bregma.

To bilaterally inject the frontal cortex, add 1.6 mm to the recorded rostral caudal bregma coordinate, and 2.6 mm to the recorded medial lateral bregma coordinate. Mark the injection sites on the skull and then drill small holes at the injection sites. With a cotton tip applicator, wipe away any excess blood or bone fragments.

Bring the needle to the surface of the brain, and lower to the depth of the dorsal ventral coordinate. Then slowly inject the contents of the syringe by twisting the plunger of the needle to administer 0.1 microliters per minute for five minutes, for a 0.5 microliter injection. Allow the syringe to remain in place for five minutes.

After the allotted time, remove the animal from the stereotaxis, close the incision, and place on a warming pad. Administer post operative analgesia according to your approved animal protocol. Perform behavioral testing two to three days after stereotactic injection of viral particles.

Habituate the mice to the room for at least four hours prior to the beginning of the experiment. Begin by dissolving DOI into a 0.9 percent saline solution to two milligrams per kilogram. Prepare a home cage without any bedding and using a tripod adjust a camera so that it is directly over the home cage.

Calibrate the camera so that the entire home cage is in the field of view. Weigh the mouse and inject intraperitoneally with the appropriate dose of either 0.9 percent saline or DOI. Place the mouse back in the home cage for ten minutes.

After ten minutes, place the mouse into the center of the empty home cage, and ensure that there are no blind spots in the field of view of the camera. Press record on the camcorder and leave the room. After 30 minutes, stop the recording and place the mouse back in the original home cage.

Repeat the behavioral test on the next mouse. Have the referee review the videos blind to the experimental conditions. Manually record every head twitch throughout the video.

A head twitch is defined as a rapid shaking head movement conducted by a mouse. Process the data as outlined in the written portion of the protocol. Shown here is a representative image of HSV mediated transgene expression in the frontal cortex.

HSV mGlu2 GFP was injected into the frontal cortex and GFP expression was revealed by immunocytochemistry. This scale bar represents 200 microns. This image shows a western blot of HSV mediated transgene expression and anti-mGlu2 reactivity in the frontal cortex of mGlu2 knockout mice.

Here we measured immunoreactivity of mGlu2 as a monomer. This histogram shows that viral mediated expression of mGlu2, but not of chimeric mGlu2 in the frontal cortex of mGlu2 knockout mice significantly rescues the head twitch response induced by the hallucinogenic seratonin 2a receptor agonist DOI. The most critical steps with this experiment are the timing between the viral injections, the head twitch response, and when the mice are sacrificed.

Any delay can alter the results of the experiment or introduce ambiguity into the data. Once mastered, the mouse surgery will take approximately 30 minutes to inject the virus. The head twitch experiment should also take approximately 35 minutes per mouse.

Staggering surgeries, or operating on more than one mouse at a time is recommended. This way one can maintain a timeline that allows the head twitch experiments to be done in the three to four days after surgery which is the peak expression time of the viral construct. Thanks for watching and good luck with your experiment.