Implantation of an Electrode Assembly in a Mouse Brain for Electrophysiological Recording

Secure an anesthetized mouse on a stereotaxic frame containing a heating pad to maintain body temperature.

Apply lubricant to prevent the eyes from drying and inject an electrolyte solution subcutaneously to maintain hydration.

Remove the fur from the scalp, disinfect the area, and inject an analgesic subcutaneously to reduce pain.

Make an incision to expose the skull, then clean it to make anatomical landmarks visible for identifying brain regions.

Apply dental adhesive and harden it using a curing light to aid in electrode implantation.

Drill holes above the hippocampus and cerebellum, and mount an electrode assembly using a holder on the stereotaxic frame.

The bipolar electrodes record hippocampal activity, while a reference electrode obtains baseline activity from the cerebellum.

Insert the electrodes, secure the assembly with dental cement, and detach from the holder.

Administer the electrolyte solution and an analgesic again.

Allow the mouse to completely recover before performing electrophysiological recording.

Before proceeding with the surgery, record the weight of the 8-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 inhalant 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 toe. 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 and 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 boreholes bilaterally into the skull at 5,000 rotations per minute, or RPM, avoiding drilling into the brain. Apply a drop of saline to enhance the drilling of the burr holes. For implantation of hippocampal depth electrodes, drill at 3 millimeters posterior and 3 millimeters lateral from bregma.

Drill one burr hole for the reference electrode above the cerebellum, behind the lambda at 6 millimeters posterior and 0 millimeters lateral from bregma. Next, mount the six-pin pedestal with assembled electrodes into the electrode holder on a stereotactic 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. Prepare 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 stereotactic arm, raise arm, and remove the holder from the pedestal. After administering the mouse subcutaneously with ketoprofen and normosol, remove the animal from the stereotactic 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.