Optogenetic Manipulation of Neural Circuits for the Sleep-to-Wakefulness Transition in Mice

Secure an anesthetized mouse with an exposed skull in a stereotaxic frame.

The brain's bed nucleus of the stria terminalis (BNST) contains virally transduced neurons expressing light-sensitive cation channels.

Drill holes in the skull, insert optic fibers, and secure them with supporting screws and dental cement.

Drill additional holes to position an assembly containing electroencephalography (EEG) and electromyography (EMG) electrodes and secure it with adhesive.

Expose the neck muscles to insert the EMG electrode wires, then allow recovery.

Connect the mouse to a recording system, place it in an experimental chamber, and allow it to sleep.

Deliver light through the optic fiber to activate the light-sensitive cation channels, triggering cation influx that generates action potentials.

The signals propagate to sleep-regulatory regions and promote wakefulness.

Record the EEG signals to monitor brain activity and EMG signals to detect muscle activity to observe the sleep-to-wakefulness transition.

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.