Combining Imaging and Electrophysiology to Visualize and Record Spreading Depolarizations in Mice

The scope of our research is to understand the relationship between spreading depolarizations and mild traumatic brain injuries using brain models. Our group and others have recently shown that spreading depolarizations occur in mild closed-skull injuries in mice. Commonly used in vivo electrophysiology amplifiers will filter out the slow extracellular shifts associated with spreading depolarizations, making it nearly impossible to detect the spreading depolarization.

The advantage of this protocol is to leverage both imaging and in vivo electrophysiology to confirm the absence or presence of spreading depolarizations following a mild traumatic brain injury. We have begun to understand the role of spreading depolarizations in the acute behavior, pathology, and long-term cognitive deficits associated with MTBIs. To begin, place the anesthetized animal in a stereotaxic frame and maintain the animal's body temperature using a heating pad.

Using a number 10 scalpel blade, make an incision along the midline from rostral to caudal. And immediately apply cyanoacrylate glue to the incision. Then, spread the skin laterally to expose the skull.

Add an additional drop or two of cyanoacrylate glue over the skull to provide extra protection. While the glue is drying, use sterile surgical scissors to increase the incision size over the cerebellum. Next, using a 0.8-millimeter drill bit, drill pilot holes through the skull and turn the screws into the skull.

Wind excess wire around the screw, allowing some slack for head stage placement later. Once all skull screws are in place, plug all the gold pins into the desired head stage connector. Holding the connector in place with the stereotaxic arm, position the connector pins close above the screw heads with no wire contact.

Seal the entire area with cyanoacrylate glue, ensuring the imaging area in front of the skull screws is undisturbed. Build up dental cement around the base of the gold pins, ensuring the gold pins from the skull screws are not connected to those in the head stage. Allow the dental cement to cure before removing the head stage connector.

To begin, turn on the IOS computer and LED panel board. Mount the electrode-implanted anesthetized animal in an impact bed beneath the camera. And adjust the focus ring to cover the cortical surface.

After loading the intrinsic optical signal, or IOS settings, press the Live button to view the mouse and center the animal's skull under the camera. Then, press Multi-D Acquisition to access the acquisition settings and select the desired time points. Save images to the designated file path with the appropriate name prefix.

Connect the gold pins from the animal to the head stage connector and then to the head stage. Turn on the electrophysiology computer and connect the computer to the electrophysiology equipment. After loading the settings for Intan software, click on the Choose File Format icon, and in the pop-up window, select One File Per Signal Type Format.

Then, in the BW tab under Hardware Bandwidth, select the amplifier bandwidth of 0.13 to 500 hertz. Select Wide under Filter Display Selector. After setting the low and high pass filters, click on the Select Filename icon to select the desired file path for saving.

For the impact, turn on the electromagnetic impactor and flick the lever to check if the tip retracts and extends properly. Quickly place the animal on a foam bed and slide the bed beneath the tip of the impactor. Align the impactor tip with the midline and position it one millimeter rostral of bregma.

Then, lower the impactor tip until it firmly touches the top of the animal's head. Push the lever to retract the impactor tip, then manually lower it by five millimeters before activating the impact lever. Immediately remove the animal from the foam bed and place it beneath the camera for imaging within five seconds.

Click on Acquire to start IOS imaging in the Multi-Dimensional Acquisition window. Capture images for four minutes to record the entire spreading depolarization event. To start the electrophysiology recording, click on the Record icon in the Intan software.

Upon completion of the recording, unplug the animal from the head stage and connector and place it back into its cage. To open the image stack, click on File, followed by Import and Image Sequence. Draw a square using the cursor, defining the region of interest, to generate the plot of the spreading depolarization.

Navigate to Image, followed by Stacks, and Plot Z-axis Profile to plot the graph and click the Live button. On the generated plot, click on List to generate a list of values. For visualization, convert the grayscale image with a fire lookup table.

Spreading depolarizations were observed propagating across the cortex and large blood vessels through the window. High frequency activity in the electrophysiology recordings showed a quick return in sham animals after isoflurane removal. But in depolarized animals, high frequency activity remained suppressed for several minutes before recovery.

The power plots demonstrated a clear rise in power after recovery from suppression in both conditions.