A Cost-Effective and Minimally Invasive Protocol for Chronic Multi-Site Electroencephalography Recording in Freely Moving Mice

This protocol enables cost-efficient, minimally-invasive, high-quality mouse EEG recordings, making long-term neural monitoring accessible for preclinical research. The major challenges of chronic EEG recording are implant instability and head cap longevity, signal degradation, massive data analysis, and inaccessibility of expensive commercialized systems. To begin, use a USB type B female plug with five pins and solder it with a 0.003-inch platinum iridium wire.

Do not use the fourth pin, as it is the ground pin, and instead use the other four pins to connect the electrodes. Bend the pins by approximately 45 degrees and wrap the wires around each pin twice to ensure a strong connection that will not come off. Now, apply a small amount of solder resin flux paste to each pin to clean the surface for effective soldering.

Then, place a small piece of solder wire on each pin. Using a soldering iron, melt the solder wire to permanently attach the platinum iridium wire to the pins. After confirming deep anesthesia, start the isoflurane oxygen ventilator on the nose at a rate of 1.5%volume per volume.

Now, inject the mouse with carprofen at five milligrams per kilogram to reduce inflammation and pain. Inject dexamethasone at 0.1 milligrams per kilogram intramuscularly to reduce inflammation and brain swelling. Apply eye ointment to prevent the eyes from drying during anesthesia.

After removing the fur, clean the incision side on the skull with 70%ethanol, followed by iodine pads while following a septic technique. Next, use a scalpel to make a single clear incision in the middle of the head. Cut around the incision to expose the site of electrode implantation, ensuring that the muscles are not exposed.

Wash the skull surface with 0.9%sodium chloride solution. Dry the surface and scrub the skull with cotton swabs dipped in hydrogen peroxide. Use a scalpel to remove any remaining connective tissue on the bone.

After one minute, wash the surface again with 0.9%sodium chloride solution and dry it before drilling. Use a dental drill to roughen the skull surface to promote acrylic adhesion. Measure and mark the electrode implantation sites at anterior/posterior positions, plus two and minus four, and medial lateral positions, plus or minus 1.5.

Drill the surface of the skull with 1.4-millimeter drills and insert four bone screws of 1.5 millimeters into the drilled sites using precision screwdrivers. Ensure the screws touch only the surface and do not enter the brain. Wrap the wires from the USB connector around each screw two to four times and cut off any excess wire.

Apply a small drop of super glue on the top of each screw and let it dry to strengthen the connection. Ensure that the connector is positioned in the middle of the skull without touching the skull or electrodes. Then, slowly build the head cap using cyanoacrylate, applying thin layers to prevent overheating of the bone.

Using dental cement, attach a large screw to the flat surface of the connector to facilitate USB cable plugging. Allow the head cap to dry for at least two minutes before removing the mouse from the stereotaxic stage. Place the mouse on a heating pad and observe it for 24 hours.

Treat the mouse with carprofen at five milligrams per kilogram every 12 hours for seven days. Wait for two weeks to let the bone grow at the acrylic interface before proceeding with electroencephalography or EEG recording. Record the EEG signal of each mouse for one hour, including 30 minutes before pentylenetetrazole treatment and 30 minutes after administering 20 milligrams per kilogram pentylenetetrazole.

Plug the USB cable into the mouse's connector. Attach the USB cable to a differential alternating current amplifier using alligator clips. Set the gain to 10, 000 and filter between 0.1 and 250 hertz.

Sample the electroencephalography signal at 1, 000 hertz. Record the amplifier outputs simultaneously on the computer and display the signals in real time on the oscilloscope. Connect the amplifier outputs to a data acquisition and signal conditioning USB device attached to the computer.

Finally, record the EEG signal using the Python code and filter it between 0.1 hertz and 250 hertz and visualize the data. Electrodes were implanted in 20 C57BL/6J background mice, including five wild-type mice with a postoperative survival rate of 85%maintained up to six months after surgery. EEG recordings captured pentylenetetrazole-induced electrophysiological changes, as shown by an increased number of interictal discharges in the mice following pentylenetetrazole injection.

The amplitude of interictal discharges after pentylenetetrazole injection was visibly higher across three different electrodes as shown in representative EEG traces. Our study established a simple stable EEG system in freely-moving mice, showing reliable long-term recordings at the clear serial-related discharges. This protocol advances neuroscience by providing accessible long-term EEG recordings in freely-moving rodents, supporting broader studies of brain disorders and therapies.

Future research will focus on refining electrode design, expanding multi-region recordings, and integrating EEG with behavioral, pharmacological, and genetic manipulations.