This protocol generates a new model for temporal lobe epilepsy in which mice developed spontaneous seizures, and it's been very useful for probing the mechanisms of epileptogenesis and also for screening novel therapies. This procedure in VGAT-Cre mice reliably leads to spontaneous recurring electrographic and motor and motor composite seizures without causing extensive hippocampal cell death that is often seen after status epilepticus triggered with chemoconvulsants or continuous electrical stimulation. To start making headsets, cut 3.5-centimeter-long polytetrafluoroethylene-coated stainless steel wire and strip about one millimeter of the insulation coat off both ends of the wire.
Put two pins on a vice holder, with the bottom part of the pin having a longer slit facing down. Then, apply flux onto the stripped ends of the wire and the tops of the pins. Tin the stripped part of the wire and the top of the pins with just enough solder to coat it without overflowing onto the sides.
Place one of the stripped ends of the wire into the pin as deep as possible while the solder is melting. Repeat the process for the second pin with the other stripped end of the wire. After cooling the pins and solder for 10 seconds, remove the pins from the vice holder and then pull onto the pins to ensure that the connection between the wire and the pins is strong and holds.
Rinse the pins in cold water followed by drying. Later, use an ohmmeter to verify conductance between pin one and pin two. Bring the pins at the end of the wire together to hold them parallel and close.
Clamp a hemostat to the center of the wire and rotate the hemostat to twist the wire around itself tightly. After removing the hemostat, clamp the forceps onto the twisted wire at two millimeters below the pins to bend the wire at 90 degrees. Push the wire 90 degrees back over the forceps, creating one more bend at one millimeter from the first.
Use small, sharp scissors to cut the twisted wire at 45 degrees and 3.5 millimeters below the bend. Prepare two bipolar or double-pin twisted electrodes for each headset. To prepare one reference electrode, cut a wire with pin soldered on both ends into two.
Then, cut the wire at seven millimeters and bend the end of the wire at one millimeter below the tip. Cover the bent one-millimeter tip of the wire with forceps and rotate the wire tight around the forceps to create a small loop with one millimeter in diameter. Then, bend the loop perpendicular to the straight part of the wire to make the wire tip point outward again.
Assemble all the electrodes into the six-pin pedestal side by side with a six-millimeter distance between the two bipolar electrodes. Place the reference electrode in the middle outer hole. Before proceeding with the surgery, record the weight of the eight-week-old VGAT-Cre mice.
Then, place the animal in an induction chamber to induce anesthesia. Place the anesthetized mouse on a heating pad at 37 degrees Celsius. If using a feedback-controlled temperature system, insert the temperature probe into the rectum of the animal for temperature monitoring during the surgery.
To start the surgery, redirect the inhalent anesthesia flow to the nose cone and mount the animal in the stereotaxic frame by gently placing the front upper teeth into the incisor bar and move the cone onto its nose. Then, place the ear bars into the ears and affix to the stereotaxic frame. Ensure that the animal's head is leveled and centered and cannot be moved when slightly probed.
Once the animal is mounted, inject 0.5 milliliters of Normosol onto the mice for hydration and apply ocular lubricant to prevent corneal drying. Monitor the depth of anesthesia by pinching a hind limb paw. If the withdrawal reflex is absent in the animal, proceed to remove the scalp hair of the mouse around the surgical area.
When done, disinfect the clean skin area thrice with an alternating application of ethanol and iodine, finishing with iodine, and inject 0.05 milliliters of 0.25%bupivacaine subcutaneously. Make an incision on the skull using a scalpel and then cut out a part of the skin with surgical scissors, exposing the skull. Push the skin aside before cleaning the skull from all underlying tissues with a cotton swab.
Clean the skull with hydrogen peroxide using sterile cotton swabs and make the skull sutures, bregma, and lambda visible. With the stereotaxically-mounted drill, move the drill tip onto bregma in zero X, Y, and Z coordinates. Then, raise and move the drill to lambda to determine its coordinates to determine if the head is level.
After drying the skull thoroughly, apply one drop of self-etching dental adhesive with the applicator. Wait 60 seconds before curing the dental adhesive with a dental UV light for 40 seconds. A glossy surface indicates that the adhesive has been effectively cross-linked with the skull.
With the help of a 0.031-inch drill bit, carefully drill two burr holes bilaterally into the skull at 5, 000 rotations per minute, or RPM, avoiding drilling into the brain. Apply drop of saline to enhance the drilling of the burr holes. For implantation of hippocampal-depth electrodes, drill at three millimeters posterior and three millimeters lateral from bregma.
Drill one burr hole for the reference electrode above the cerebellum behind the lambda at six millimeters posterior and zero millimeters lateral from bregma. Next, mount the six-pin pedestal with assembled electrodes into the electrode holder on the stereotaxic frame. By aligning the electrodes above the corresponding burr holes, slowly lower and navigate the headset to locate the two bipolar electrode tips right on top of the left and the right hippocampal burr holes, and if needed, adjust the reference electrode to hover above hole over the cerebellum.
When the hippocampal twist electrodes are right above the holes, zero the Z axis and slowly lower the electrode to minus three millimeters into the right and left hippocampus and guide the reference electrode into the hole above the cerebellum. Prepared the dental cement by mixing the powder and liquid in a mixing bowl. Then, cover the skull surface, the electrodes, and the space between the skull surface and the bottom of the pedestal with dental cement.
Once the cement dries and hardens, detach the electrode holder from the stereotaxic arm, raise arm, and remove the holder from the pedestal. After administering the mouse subcutaneously with ketoprofen and Normosol, remove the animal from the stereotaxic frame to move the animal onto the heated pad in the vivarium cage. Once fully awake, return the single animal to the vivarium cage, feed the animal with soft food, and monitor the weight loss daily up to 72 hours post-surgery.
In the representative violin plot, the number of stimulations required by mice to the kindled state criterion is shown. The mice typically reached the kindled state or experiences five bilateral tonic-clonic motor seizures after 15 stimulations. The latency to spontaneous recurring seizures, or SRS, was found to be 10.7, and the average frequency of the seizures was 1.3 seizures per day.
The reliable SRS distribution showed that the mice were epileptic for approximately 23 days. In the representative images, the number of anti-NeuN positive neurons or neuronal deaths in various subfields of the hippocampus and entorhinal cortex can be seen. It is important to have the two bipolar electrodes smoothly enter the two burr holes, so they need to be right above the burr holes.
And also, it's important to carefully apply the dental cement and let it harden completely before removing the electrode holder. This technique allows us to generate epileptic mice and continuously monitor them with EEG recording and also video recording, simultaneously. And with the experiments we do, we can then test novel treatments strategies such as gene therapy to reduce the frequency, the duration, and the severity of the seizures.
