Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

All experiments here were approved by the Animal Experiment and Use Committee of the University of Tsukuba, complying with NIH guidelines.

1. Animal Surgery, Virus Injection, Electrode for EEG/EMG, and Optical Fiber Implantation

CAUTION: Appropriate protection and handling techniques should be selected based on the biosafety level of the virus to be used. AAV should be used in an isolated P1A graded room for injection, and the tube carrying AAV must be sterilized with an autoclave after all the volume is used up. The surgical site and all implanted material should be clean and sterile during use.
NOTE: See Figure 1.

1. Disinfect the surgical equipment with the autoclave.
2. Anesthetize mice with isoflurane using an anesthetic vaporizer. Observe until the mouse has reached the desired depth of anesthesia, determined by loss of response to pinching the tail with forceps. Apply ophthalmic ointment to the eyes to prevent them drying.
3. Disinfect the surgical field with iodine solution or 70% EtOH (3x) and dry sufficiently. Allow the virus to thaw on ice as the surgery is being performed. Cover the surgical area with absorbent lab bench paper.
4. Fix the mouse's head in the stereotactic apparatus with ear bars and a nose pinch. After confirming the head is held stably, make a midsagittal incision in the scalp to ensure the positions of the bregma and lambda are located at the same level on a horizontal line.
5. To avoid a positioning gap, appropriately adjust the levels of the nose pinch and ear bars up and down. The bregma and lambda refer to the intersection between the sutura saggitalis and sutura coronalis or sutura lambdoidal, respectively (Figure 2).
6. Use Serafin clamps to hold the skin to maintain access to the cranium. After exposure of the skull, disinfect the surface of the skull with iodine or 5% H₂O₂, to enable the cranial sutures including the bregma and lambda to be visualized more clearly.
7. Prepare AAV vector injection:
   1. Wash the inside of a 10 mL syringe (see Table of Materials) sequentially with 70% EtOH, 100% EtOH, and sterilized water, 5 times each. Secure the syringe in the clamp of a microinjection pump arm and make sure all solution in the syringe is discharged.
   2. Carefully aspirate 2 µL of mineral oil without air bubbles, then aspirate the designated volume of the virus solution. After aspiration, manipulate the plunger button and confirm that virus solution emerges at the tip of the needle.
      NOTE: The injection volume of the virus solution was determined in pilot experiments using the same mouse strain and same virus product. The relation between the volume of virus solution and extent of the infection area should be estimated in advance.
8. Inject AAV vector:
   1. Adjust the tip of the microinjection needle on the bregma and note the coordinates as the original point. Move the tip to the designated injection site (for the BNST: anteroposterior + 0.2 mm, mediolateral ± 1.0 mm, dorsoventral – 4.2 mm) and place the tip of the needle onto the position. Put a mark on the skull and drill holes of approximately 2 mm in diameter using a dental drill with a 0.7 mm carbide cutter. Be careful not to damage the dura or brain tissue.
   2. After removing blood from around the holes with a cotton swab, slowly move the needle into the position of the BNST. Slowly inject the designated amount of virus solution (0.07 µl/min) with a mechanical microinjector. After completing the injection, leave the needle for 5 min to allow the solution to sufficiently infiltrate the BNST tissue. Carefully take out the needle.
   3. For bilateral injection, repeat steps 1.8.1-1.8.2 on the other side. Throughout the procedure, keep the skull moist with applications of sterile saline.
      NOTE: We used custom EEG/EMG implants (W: 5 mm, D: 7 mm, H: 1 mm) with four EEG electrodes (4 mm), two EMG electrodes (2 mm; cut the 4 mm electrode to 2 mm with nippers) and 6 electrodes (4.5 mm) (Figure 2A).
9. Solder two stainless steel wires (see Table of Materials) from which 1 mm of the insulation is stripped off both ends to the EMG electrodes. Adjust the center of the electrodes to the bregma and mark the position of each EEG electrode (anteroposterior ± 1.5 mm, mediolateral ± 1.0 mm), and determine the position of the implant (Figure 2B).
10. Implant optical fibers:
    1. Attach an optic fiber ferrule to the manipulator and rotate the manipulator arm so that it has an angle of ±30° against a horizontal line (this process is only needed to avoid interference between electrodes and optic fibers, such as in the case of BNST stimulation). Put the fiber tip on the bregma and record the coordinates.
    2. Move the tip to the targeted insertion line and mark the position on the skull. Also put additional marks near the insertion site for the anchor screws. Drill the skull on each site with a dental drill to insert the optic fiber and fix the screw. Fix the screw on the skull. Be careful not to break the dura or damage any tissue by screw.
    3. Insert the optic fiber gently until reaching above the BNST with a manipulator. The ferrule should rest on the remaining cranium (Figure 1B).
    4. Apply photocurable dental cement (see Table of Materials) to cover the fiber and the screw. The reaction time to solidify the glue should be specified by the manufacturer's manual (Our material needs exposure to light for at least 10 s with specific wave length photo-generator. It is unnecessary to dry the glue after this).
    5. In this step, make sure that no materials (screw or glue) occupy the mounting space for the electrodes. In addition, avoid making any interruption in the cement for the ferrule connecting to the optic fiber and cable. Repeat steps 1.10.1-1.10.4 on the opposite side for bilateral stimulation.
11. Drill holes for EEG/EMG electrodes. Insert the tips of the electrodes into the holes. Hold the implant and apply cyanoacrylate adhesive to the space between the skull and the electrodes. Insert again with attention not to interfere with any materials.
12. Cover the circumference of the electrodes and optic fibers with cyanoacrylate adhesive followed by application of cyanoacrylate accelerant on the adhesive. This step avoids causing any interruption at the ferrule-to-optic cable and electrode-to-lead wire connecting zone (Figure 1C).
    NOTE: Cyanoacrylate adhesive and its accelerant are harmful to the mouse eye. Pay attention not to cause spillage of these chemical substances. Also, be careful not to strongly touch the electrodes and the fibers in order to avoid unexpected deviation immediately after adhesive solidification.
13. Expose the mouse neck muscles and insert the wires for the EMG electrode under the muscle. Adjust the length of EMG electrode so that it locates just under the nuchal muscles. Light connection between tip of electrode and muscle fascia is enough to catch the EMG signal.
14. Apply cyanoacrylate adhesive to fill the implants and solidify the adhesive with acceleration liquid. Then, put the mouse on a heat pad for recovery until the postural reflex appears. Adjust heat pad temperature to animal resting body temperature (36.0 °C in ZT 0-12 in case of C57BL6 mice; do not exceed 38.0 °C).
    NOTE: An antibiotic is not required for sterile surgery.
15. Follow your local institutional guidelines for post-operative analgesia. Keep the mice in a home cage for a recovery period of at least 7 days.

2. EEG/EMG Monitoring with Photo-excitation of Targeted Neurons in Specific Sleep States

CAUTION: This protocol includes use of class 3B laser equipment or LED devices. Experimenters should be aware of safety information. Protective eye goggles are required.

1. Before connecting the laser cable to the optic fiber, adjust laser intensity with a scaler. Tether the tip of the laser cable to an unused optic fiber with a ferrule and confirm that there is no space at the junction between the fiber and the cable.
2. Turn on the main switch of the laser and wait 20 min for it to warm up.
3. Emit the laser to the intensity checker and adjust laser intensity to 10 mW/mm². Change the laser mode to transistor logic and confirm that light pulses are emitted from the fiber controlled by the pattern regulator which is set at 10 ms for duration, 40 ms for rest, 20 times for cycle, and 20 times repeat (that is, 20 Hz of 10 ms light pulses for 20 s).
4. After the recovery period, move the mice to the experimental chamber for recording EEG/EMG. House mice at a constant 23 °C with a 12 h light/dark cycle with food and water available ad libitum.
5. Connect the implanted electrode and cable adaptor which is tethered to a slip ring to avoid entanglement. It is recommended to cover the junction with light-impermeable material such as aluminum foil to prevent laser leakage. If a bilateral experiment is required, use a slip ring with a bifurcate attachment for the cables.
6. In this protocol, we assess latency to wakefulness from NREM sleep or REM sleep, so the recording time should be limited in optimized zeitgeber time (ZT0 is defined as the time when the light is on). This protocol was conducted between ZT4 – ZT10. Let the mice stay freely in the experimental chamber for at least 1 h as acclimatization.
7. During the experimental period, monitor EEG and EMG signals in the same monitor and evaluate the mouse's state as wakefulness, NREM sleep or REM sleep. Use the gain control for each wave to make it easier to distinguish each state.
8. For measurement of NREM sleep to wakefulness latency, observe stable NREM sleep for 40 s or stable REM sleep for 30 s, then turn on the switch of the pattern generator for photostimulation (this protocol generates 20 Hz of 10 ms light pulses for 20 s). Confirm laser emission to the implanted optic fibers.
9. Record EEG/EMG signals until the sleeping state changes to wakefulness. If two or more experimental trials are needed, limit optogenetic manipulation to once a day because photostimulation is an artificial intervention which might affect sleep/wakefulness architecture.
10. After the experiment, deeply anesthetize and perfuse with sterile saline and paraformaldehyde (PFA) for sampling the whole brain for immunohistochemical analysis²⁶.

3. Analysis of Latency Time from NREM Sleep to Wakefulness

1. After recording EEG/EMG signals, transfer the signal data to a computer for scoring sleep/wakefulness states. This protocol describes the method for EEG analysis with recording software (see Table of Materials).
2. Start the sleep sign application and click the File tab and select Open to choose the recorded data (.kcd file). Click the Sleep tab to select Epoch time for adjusting the time window for each epoch (we use 1 epoch/4 sec).
3. Manually score sleep/wakefulness states based on EEG/EMG signals, according to the following criteria: wakefulness, high EMG and low EEG voltage with high frequency; NREM, low EMG tone and high EEG voltage with high δ (0.5-4 Hz) frequency; and REM, EMG indicates muscle atonia and low EEG voltage with high θ (6-9 Hz) frequency. A state that does not consecutively continue for 16 s (i.e. 4 epochs) is not defined as a state change because it is not a stable state.
4. Click and hold the left mouse button on the first epoch and drag the cursor until the specific trend of more than 16 s (4 epochs) is ended. Then, release the left mouse button and choose the appropriate state (wakefulness or NREM sleep or REM sleep) in the pop-up window. Repeat this procedure to score all EEG/EMG signals recorded in the file.
5. Find the exact time of stimulation in the epoch where the EEG shows NREM or REM sleep, and the epoch showing state transition following the stimulation point. Count the number of epochs between the periods just after stimulation and just before state transition.
6. Then multiply counted number of epochs by 4 s (A). In the epoch of stimulation, take a screen shot and measure the width between the stimulation point and the end of the epoch. Then, divide the measured length by the entire epoch length and multiply by 4 s (B). Similarly, calculate the duration of NREM sleep in the epoch of state change, which means the time between the start of the epoch and the state change point (C).
7. Sum A, B and C to obtain the latency from NREM sleep to wakefulness. The same procedure is used for analysis of state transition from REM sleep to wakefulness.