This procedure can help us identify the function of specific neuro circuit that are involved in the sleep wakefulness regulation with less invasive manipulation and high temporal resolution. This method allows us the monitoring surface EEG in real time in conjunction with manipulation of a specific neuro circuit to examine the causal relationship between circuits and sleep wakefulness states. Shota Kodani is going to demonstrate the procedure.
After confirming a lack of response to toe pinch, apply ointment to the animal's eyes and use the ear bars and nose pin to the stereotactic apparatus to stabilize the head of the anesthetized mouse on an absorbent pad. When the head is fixed in position, make a mid-sagittal incision in the scalp to confirm that the intersection between the sutura sagittalis and sutura coronalis or sutura lambdoidea are in a horizontal line at the same level. To avoid a positioning gap, adjust the nose pinch and ear bars as necessary.
Then using serafin clamps to the hold the skin open disinfect the exposed surface of the skull to enable the cranial sutures to be visualized more clearly. To inject the adeno-associated virus vector, load an ethanol sterilized 10 milliliter syringe with two microliters of mineral oil, followed by the experimental volume of virus to be injected. Use the microinjection pump to adjust the tip of the microinjection needle onto the bregma and note the coordinates as the point of origin.
Then move the tip to the designated injection site and place the tip of the needle onto the position. Mark the skull at the injection site and use a dental drill equipped with a 0.7 millimeter carbide cutter to drill an approximately two millimeter diameter hole into the skull. After removing blood from around the hole with a cotton swab, slowly move the needle to the bed nucleus of the stria terminalis or BSNT and slowly inject the designated volume of virus solution.
Then leave the needle in place for five minutes to allow the solution to sufficiently infiltrate the BNST tissue before carefully retracting the needle. For electroencephalogram, or EEG, and electromyogram, or EMG, electrode implantation, first solder two stainless steel wires from which one millimeter of insulation has been stripped from both ends to the EMG electrodes and place the center of the electrodes onto the bregma. Then mark the position for each EEG electrode.
To determine the position of the optical fiber implant, attach an optic fiber ferrule to the manipulator and rotate the manipulator arm to a plus or minus 30 degree angle against a horizontal line. Put the fiber tip on the bregma and record the coordinates. Move the tip to the targeted insertion line and mark the position on the skull and the position for the anchor screw next to the insertion site.
Use the dental drill to drill the skull at each site and use the manipulator to gently insert the optic fiber until it reaches above the BNST. The ferrule should rest on the remaining cranium. Secure the fiber to the skull with an anchor screw, taking care not to break the dura or damage any tissue.
Then cover the fiber and screw with photo-curable dental cement. Next, drill holes for EEG/EMG electrodes and insert the tip of the first electrode into one hole. Holding the implant with one hand, apply cyanoacrylate adhesive to the space between the skull and the electrode and insert the electrode the rest of the way, taking care not to damage any tissue.
When all of the electrodes have been placed, cover the circumference of the electrodes and the optic fibers with additional cyanoacrylate adhesive and cyanoacrylate accelerant to avoid causing any interruption at the ferrule to optic cable and electrode to lead wire connecting zones. Now expose the neck muscles, and insert the wires for the EMG electrode under the muscle. Adjust the length of the EMG electrode so that it fits just under the nuchal muscles and fill the implants with more cyanoacrylate adhesive and accelerant.
Then place the mouse on a heat pad with monitoring until full recompensy. For EEG/EMG monitoring during the photo-excitation of targeted neurons, first use a scalar to adjust the laser intensity and use a ferrule to tether the tip of the laser cable to an unused optic fiber. Confirm that there is no space at the junction between the fiber and the cable.
After 20 minutes, emit the warmed up laser to the intensity checker and adjust the intensity to 10 milliwatts per millimeter squared. Set the light pulse duration to 10 milliseconds, the rest period to 40 milliseconds, the cycle to 20, and the repeat to 20. Change the laser mode to transistor logic and confirm that light pulses are emitted from the fiber controlled by the pattern regulator.
Connect the implanted electrode and cable adapter, then cover the junction with light impermeable material to prevent laser leakage. And when the laser is ready, move the mice to the experimental chamber for EEG/EMG recording. To assess the latency to wakefulness from non rapid eye movement or rapid eye movement sleep, limit the recording time and optimized site gain time and let the mice move freely in the experimental chamber for at least one hour.
During the experimental period, monitor the EEG and EMG signals in the same recording screen. Evaluate the mouse's state as wakefulness, non rapid eye movement sleep, or rapid eye movement sleep using the gain control for each wave for ease of distinguishing each state. For measurement of the non rapid eye movement sleep to wakefulness latency, observe stable non rapid eye movement sleep for 40 seconds, then turn on the pattern generator for photo stimulation and confirm laser emission to the implanted optic fibers.
Then record the EEG/EMG signals until the sleep state changes to wakefulness. Here represented EEG/EMG traces before and after photo stimulation during non rapid eye movement sleep are shown. A high voltage and slow frequency EEG with no EMG signals, represents non rapid eye movement sleep.
Photo stimulation triggered an acute transition to wakefulness about two seconds after stimulation in channelrhodopsin-2 expressing mice while control mice did not demonstrate this transition, suggesting that the excitation of BNST GABA neurons during non rapid eye movement sleep triggers a rapid induction of wakefulness. Conversely, photo stimulation during rapid eye movement sleep had no effect so a transition effect only emerged in non rapid eye movement sleep. It is important to take care to precise coordinations for the virus injection and optic fiber implantation steps.
The EEG/EMG traces captured during the sleep analysis can be used to assess the consequences of the photo manipulation on the different vigilant states in mice. This technique will also help us identify other components and circuits involved in regulating the sleep wakefulness states. Use protective goggles to avoid laser damage to the eye and be sure to always autoclave sterilize the adeno-associated virus vector container after use.
