Begin by transferring the anesthetized animal to the stereotaxic frame, and then secure the head into the nose cone and ear bars. Place the probes for measuring the pulse, pulse oxygen saturation, blood pressure, and temperature of the animal. Apply eye ointment.
Remove the hair with depilatory cream and aseptically clean the scalp with three passages of iodine and alcohol. For a terminal study, remove the scalp using surgical scissors and expose the skull at approximately three millimeters from the lamboid suture caudally and three millimeters frontal to the bregma. Excise the scalp parietally to expose the inner part of the temporal muscle on both sides.
Then, remove any residual subcutaneous connective tissue so that the skull is clean and dry to apply the stimulating electrodes. Next, apply conductive gel to the side of the electrodes that will be in contact with the skull. Place the electrode on this temporal muscle and secure it with surgical super glue around the edge at intermittent spots.
Then, apply lidocaine paste to the temporal muscle and scalp on both sides without disturbing the electrodes. Test the impedance of the extracranial stimulating electrodes. Then, place the mouse under a laser speckle imaging device without intracranial recording electrodes.
To apply stimulation between the two skull tACS electrodes, use a commercial human compatible stimulating device that delivers a constant current. Before stimulation, obtain a clear baseline. Apply brief periods of on/off stimulation at various amplitudes on either side of the skull.
Ensure a clear baseline after stimulation. After the extracranial stimulating electrodes are placed four millimeters laterally to each side of the skull, mark the position of the burr holes at two millimeters on each side of the midline, four millimeters apart from each other, and orthogonal to sagittal suture. Then, drill two burr holes for the glass electrodes.
Fill these burr holes with sterile mineral oil to prevent current ingress into the skull from the extracranial electrodes. Then, fill pooled glass micro electrodes with 0.2 molar sodium chloride, and place them using a micro manipulator into the two burr holes placed laterally to the sagittal suture. Place the glass micro electrodes into the burr holes.
Next, test the impedance of the extracranial stimulating electrodes after the burr hole placement to verify that these do not interfere with the current flow into the brain. To measure the cerebral blood flow resulting from extracranial stimulation, place the mouse under a laser speckle imaging device with intracranial recording electrodes. Once inserted into the brain, perform depth profiles at various symmetrical depths to ensure these glass micro electrodes are approximately one millimeter within the cerebral cortex.
Use a digitizing system to record continuous data from the Doppler probes and micro electrodes over a sufficiently stable baseline duration. Then, apply on/off stimulation and test the extra cranial stimulation. For experiments to induce spreading depression, add a third burr hole on the right side of the skull, 1.5 millimeters rostral to coronal suture, and one millimeter lateral to posterior frontal suture.
Fill this burr hole with KCL for later application. In response to low intensity tACS, data was obtained for the intracranial direct DC electrical recordings and the bilateral laser Doppler recordings of the cerebral blood flow. A small response was observed in response to a 0.75 milliampere stimulus, while a larger response was seen in response to a one milliamp stimulus.
For both electrical and cerebral blood flow traces, the responses were asymmetrical between the right and left side of the skull. Laser speckle skull imaging of the cerebral blood flow during attacks showed increased blood flow in response to the stimulation, which was diffused evenly throughout the cortex. After the stimulation, the blood flow returned to baseline.
