Position the anesthetized mouse in a stereotaxic frame. Identify bregma and lambda, ensuring no more than a 100 micrometer height difference between the two landmarks. Find and mark the calculated coordinates on the skull with a sterile pencil.
Around the coordinates, create an outline of the two millimeter by two millimeter craniotomy window. After checking the depth of anesthesia, use a high-speed drill to create a two millimeter by two millimeter craniotomy window. Apply 0.5 to 1 milliliter of normal saline to prevent the brain surface from drying.
Remove the dura using a syringe needle and fine forceps. Next, use a high-speed drill to create a separate burr hole for the silicon probes reference electrode, generally one to two millimeters from the cranial window. Apply 0.2 milliliters of low toxicity silicon adhesive on the skull to completely seal the craniotomy.
Using the head plate and screws, affix the head of the mouse to the electrophysiology recording rig. Then coat the silicon probe shank with fluorescent dye so that the probe trajectory can be reconstructed after the experiment. After that, mount the probe on the manipulator and set the desired angle.
To lower the recording probe to the brain surface within the center of the cranial window, manually insert the probe to a depth of approximately 300 micrometers. Once inserted to this depth, slowly lower the probe automatically to 200 micrometers per minute to the targeted depth to minimize tissue damage. Finally, apply mineral oil to the brain surface within the craniotomy window to prevent drying.
Then record data from the silicon probe and ECOG at 30 kilohertz using an Intan recording controller. Most recorded neurons decreased their firing during sevoflurane anesthesia. VLPAG firing significantly decreased from baseline to during sevoflurane.
This decrease was consistent across all VLPAG neurons.
