Place the anesthetized mouse in a stereotaxic frame over a heating pad and apply ophthalmic ointment to the eyelids to prevent the eyeballs from drying. Shave the head in the area from the nasal bones to the occipital bones, and disinfect the exposed skin with alternating rounds of chlorhexidine and alcohol three times each. Using straight dissecting scissors, cut off the scalp, hold it with micro forceps, and clean the skull from fascia.
Using a drill with a 1.3 millimeter diameter, make two holes in the temporal bones on each side along the coordinates for the two screw pairs. Place the electroencephalographic, or EEG screws, with wire leads in alcohol for 15 minutes, followed by the saline solution. Place four silver plated screws with electrodes into the holes to a depth of one millimeter.
Fix the screws on the surface of the skull using dental acrylic, followed by attaching an EEG recording sensor to the animal's nose. After 30 minutes, use curved tweezers to fix the EMG electrodes on the back of the orbicularis oculi muscle with dental acrylic. Connect and fix the EEG electrodes to the silver-plated recess of the sensor.
Finally, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia. After seven days, use dental acrylic and Dumont forceps to fix a metal plate with a diameter of five millimeters on the occipital bone of the mouse skull. To prepare the chronic catheter, mark a segment on the insulin needle two millimeters from the side of its beveled end.
Fix the insulin needle in the holder from the side of the beveled tip to the marked segment. Place a two-centimeter PE-10 polyethylene catheter over the entire remaining length of the needle. Fill the catheter with a saline solution and cover it with a plastic cap.
For the implantation, drill a 1.5 millimeter diameter trepanation hole at the appropriate coordinates. Place the PE-10 polyethylene catheter in a stereotactic holder and insert it into the mouse's skull. Afterward, fix it with dental acrylic and let it harden for 15 minutes.
Connect any commercial EEG recording system to the connector on the mouse's head, and set the requirement value of the pulse width modulation, or PWM duty cycle. Monitor the EEG signal and wait for delta rhythm activity. If non-rapid eye movement, or NREM, sleep is seen, initiate the photobiomodulation, or PBM process and stop the process if NREM sleep transitions to rapid eye movement sleep or wakefulness.
