Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

The overall goal of this procedure is to allow transcranial imaging, a fluorescently labeled synaptic structures in the living mouse cortex by two photon microscopy. The first step after anesthetizing the mouse is to perform a thinned skull preparation. Next, the head of the mouse is immobilized using a custom made holding plate.

Then image stacks are acquired through the thinned skull using two photon microscopy. Ultimately, relocating and imaging. The previously imaged region allowed the dynamics of dendritic spines to be traced over different periods of time under various conditions.

To prepare for surgery, anesthetize a mouse with ketamine and xylazine and perform a toe pinch to ensure complete anesthesia. Then place the mouse on a heating pad and use a razor blade to shave the head. Wipe the skin with alternating alcohol pads to sterilize the shaved area and remove any residual hair clippings.

Also, gently apply eye ointment to both eyes. Next, make a straight incision along the midline of the scalp and move the skin laterally towards the edges of the skull. Then remove the connective tissue attached to the skull to begin the thinned skull preparation.

First, identify the desired imaging region based on its relative location to the midline and bgma. Then moisten the skull with saline to observe the vasculature so as to avoid thinning the skull in an area with large blood vessels as they block light penetration and blur the image structures. Once an area of 0.5 to one millimeter diameter has been selected, use a high speed micro drill to thin a circular region of the skull.

Move the drill parallel to the skull surface rather than holding it against the skull and pressing down to prevent damage caused by overheating. Avoid prolonged contact between the drill bit and the skull drill until both the outer compact bone layer and the middle spongy bone layer are removed. Next, use a microsurgical blade held at approximately 45 degrees to thin the inner compact bone layer in the center of the drill thinned region.

Scrape the skull without pressing down against the skull until an evenly thinned region of 200 to 300 micrometers diameter with a thickness of approximately 20 to 30 micrometers is obtained. Once the skull is thinned sufficiently, carefully remove the bone debris. The next step is to attach the head plate, which is fashioned from razor blades with sharp edges covered by tapes.

Begin by placing a small drop of cyanoacrylate glue on each edge of the center opening of the head plate. Then centering the head plate on the thinned region. Hold it tightly against the skull.

Now, gently pull the skin from the sides of the skull to the edges of the center opening of the head plate. Hold the head plate in place for one to two minutes. Then wait another eight to nine minutes until the head plate is well attached to the skull.

After 10 minutes, place the head plate on the two lateral blocks of the holding plate. Next, tighten the screws over the edges of the head plate to immobilize the mouse on the holding plate. Look at the mouse's head through a dissecting microscope while gently padding the mouse's back to be sure that the head is properly immobilized.

Next, rinsey exposed skull with saline to remove any un polymerized glue in order to relocate the previously imaged region. In subsequent imaging sessions, take a photo of the vasculature of the thinned region of the exposed skull, which is designated map one. Then place the mouse under the imaging microscope and locate the thinned area under the 10 x air objective.

Using epi fluorescence, move the thinnest area to the center of the view. Now add a drop of saline to the top of the skull and switch to the 60 x objective. Select a region where individual dendritic spines are clearly visualized along dendrites.

Identify and label the corresponding region on map one by comparing vasculature between the epi fluorescent view and the vasculature photo. Next, tune the two photon laser wavelength according to the fluoro fours. Then using the 60 x objective acquire image stacks with two micrometer steps along the Z axis, covering an approximately 200 micrometer by 200 micrometer area.

This is designated as map two and is used for relocating the image dendritic segment during subsequent imaging sessions. Now acquire nine image stacks within map two using three x digital zoom. Each image stack covers an approximate area of 70 micrometer by 70 micrometer with 0.7 micrometer steps along the Z axis.

Following imaging, gently detach the head plate from the skull. Next, use a microsurgical blade to remove the remaining glue from the skull. Then rinse the skull and the skin with saline several times and suture the scalp with sterile surgical sutures.

During recovery, keep the animal on a heating pad in a separate cage and administer buprenorphine analgesic subcutaneously for postoperative pain. After full recovery, return the animal to the home gauge To re-image dendrites on another day, anesthetize the animal and expose the skull. As before, repeat the head immobilization by mounting the head plate and attaching it to the holding plate.

Then locate the previously imaged region by comparing the vasculature pattern to map one and the dendritic branch pattern to map two. If re-imaging is performed within one week, the thin layer of newly grown bone can be removed with microsurgical blade. If re-imaging is performed after more than one week, first use the micro drill, then the microsurgical blade to thin the skull.

Now adjust the position and orientation under the two photon microscope to obtain image stacks that match the previously taken image stacks. After acquiring images, allow the mouse to recover and return it to its cage as before, and an image of the vascular pattern surrounding the thin skull area designated as map one is used to reference all subsequent imaging sessions. The black box indicates the region where two photon in vivo images were acquired.

This image of dendritic branches in the motor cortex of a one month old mouse is designated as map two. The white box shows the dendritic segment selected for imaging. Over time, two photon imaging was done of the same dendritic segment on day zero, day two, day four, and day eight.

Newly formed spines. That is spines that were not present in the previous imaging session are shown by arrowhead spines that were eliminated between imaging sessions are shown by the arrows. Philip Podia are indicated by stars once mastered.

This technique can be done in one to two hours if it is performed properly.