In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This method provides a way to monitor the circadian clock gene expression in real time in high temporal resolution in freely moving mice. The main advantage of this technique is that it works in both light-dark, and dark-dark conditions, which are critically important for circadian clock studies. Using an optical fiber knife, cut a fiber 17 millimeters in length.

Making sure that each side of the optic fiber is smooth. Insert the optic fiber into the ceramic ferrule and fix the optic fiber to a ceramic ferrule with methyl ethyl ketone peroxide. Ensuring that the surfaces of the optic fiber and ceramic ferrule are flush.

Clean the optic fiber with a cotton swab soaked with 75%ethnol. And keep it clean until ready for insertion. Begin the injection procedure by first loading five microliters of recombinant AAV into a microsyringe.

Then after anesthetizing an adult mouse using an institutionally approved protocol, check for a sufficient depth of anesthesia by the lack of a toe pinch response. Next, use scissors to remove fur from the head of the mouse. And then mount the head of the mouse into a stereotaxic apparatus.

After making an incision in the scalp, expose the skull. And adjust the stereotaxic apparatus so that bregma and lambda are in the same horizontal plane. Then use a cotton swab soaked in 4%hydrogen peroxide and water, to corrode the brain periosteum.

Followed with a clean cotton swab to clear and dry the surface of the skull. Using the stereotaxic arm, identify the surgical site, 0.46 millimeters posterior and 0.25 millimeter lateral to bregma. Then using a micro drill, make a hole, about one millimeter in diameter at this point.

Use a clear cotton swab with physiological saline solution to clear bone fragments from the exposed surface of the brain. Next, insert a microsyringe into the brain to a depth of 5.7 millimeters from the surface of the skull. And inject 500 nanoliters of recombinant AAV at a rate of 50 nanoliters per minute into the SCN.

Leave the microsyringe in place for 10 minutes after the injection to ensure complete diffusion of recombinant AAV. After 10 minutes, slowly withdraw the microsyringe. Immediately after recombinant AAV injection, insert the ceramic ferrule containing the optic fiber, into the SCN.

Then fix the ceramic ferrule and optical fiber onto the skull using dental resin and allow to dry. After drying, smear the surface of the dental resin with black nail polish. Place the mouse in a warmed recovery cage and monitor it, as it comes out of anesthesia.

Once the animal has regained a sternal recumbency, it can be single housed and returned to the housing room with a 12 hour light-dark cycle. Eight days after surgery, measure the florescent signal in the SCN of sham operated control mice to establish the background signal. First, connect the fiber on the mouse head, with the fluorescent signal monitor.

Set the software to measure the florescence for 15 seconds every 10 minutes at a frequency of 100 hertz. Record the signal from the sham injected mouse. Repeat the process to evaluate the fluorescent signal of mice injected with the recombinant AAV crypto chrome reporter.

Exclude mice which have received the viral vector, but only display background signal as this likely indicates the the tip of the optic fiber has missed the SCN. After these initial measures, return the mice to their home cages for another three weeks, to allow the fluorescent signal to stabilize. At the conclusion of the experiment, check the placement of the optic fiber in the fixed brains.

Exclude mice from analysis if the optic fiber tip is not correctly implanted in the SCN. Using the approach detailed above, 500 nanoliters of virus harboring the crypto chrome gene reporter was successfully injected into the SCN of an adult mouse. Robust venus expression was seen eight days after the virus injection.

This magnified image demonstrates the high level of venus expression in the SCN. Background florescence in the SCN for a sham operated animal during light on and light off periods is seen here. This animal showed no detectable rhythm under both light-dark and dark-dark conditions.

This image shows crypto chrome gene reporter expression over the course of seven days in a 12, 12 light-dark condition. And over seven days in a dark-dark condition. This image shows analysis of circadian rhythm in the light-dark condition.

The red line indicates the converged fit in the sin curve. The result of which is shown here. Analysis of circadian rhythm in the dark-dark condition is shown here.

Again, the red line indicates the converged fit in the sin curve. And the result of the fit is shown here.