Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

The protocol describes how to perform viral injections and implant a cranial window for imaging of brain cells in awake, head-restrained mice. The advantage of this method is that structure and activity of neurons and astrocytes can be imaged repeatedly over multiple sessions. This approach can be used to determine how neural cells are altered in models of neurodevelopmental, or neurodegenerative disorders, or whether experience-dependent plasticity is impaired.

Begin by securing an anesthetized mouse to a stereotaxic frame using the snout clamp and ear bars. Using sterile surgical tools, cut and remove the skin from the frontal sutures anterior to the bregma to posteriorly to lambda. Using a sterile size 11 carbon steel surgical blade, gently scrape all the connective tissue from the skull.

With the help of a dental drill, gradually drill the skull bone along the outline of the opening in concentric circles to facilitate even thinning of the bone. After each concentric pass, add a drop, or two of saline to the drilled area, and allow the saline to sit for at least 10 seconds before resuming the drilling. Gently push on the central area of the skull bone with fine forceps to check that the skull is adequately thinned and moves.

Ensure that the underlying vasculature where the drilling occurred is intact, and that the bone is not cracked. Carefully insert a miniature 15-degree pointed blade into the thinned bone and cut and use the gel foam soaked in saline to stop any bleeds that may occur. Using forceps, carefully lift and remove the bone, taking care to not damage the dura mater.

For the injection, fill a sterile beveled glass pipette with a 20-micrometer tip with the virus mixture. Then lower the pipette to touch the surface of the brain, and continue to lower for an additional 200 to 300 micrometers for layer 2/3 injections. Using an intracellular microinjection dispense system, pressure inject the system 12 to 15 times over two minutes.

For the cranial window implantation, place the cover glass over the opening in the skull. Use forceps to ensure that the glass is flush over the opening. To seal the edges of the glass window to the skull, apply cyanoacrylate adhesive gel around the perimeter of the glass surface.

Over the adhesive gel, apply a layer of glue. Then add a layer of dental cement liquid. When dental cement hardens, apply a thin layer of glue around the central opening of the helicopter type head plate, and place the helicopter type head plate of the appropriate size over the cover glass.

Allow the glue to dry. Add dental cement powder in a 1.5-milliliter microfuge tube up to the 0.1 milliliter mark. Mix seven to eight drops of fast-curing instant adhesive into the powder, and draw the resultant mixture into a one-milliliter syringe with a 19 gauge needle that has been cut to create a larger opening.

Inject the powder mixture through the lateral holes of the helicopter bar until it seeps from either side. Apply the dental cement adhesive mixture to the rest of the exposed skull to fasten the head plate to the skull. Wrap the mouse in cloth, and secure it via its head plate to the head fixation arm of the airlifted home cage, then leave the home cage exposed to light.

After a habituation time of 15 minutes, remove the mouse from the home cage. Using the wide field mode of the microscope, select two to three positions of easily identifiable vasculature. Save the images of the blood vessels, and record the x and y-coordinates that appear on the motor controller.

Image the synaptic structures of neurons, and GCaMP6f activity in astrocytes. In the representative analysis, the quality of the cranial window was evaluated with repeated imaging of dendrites and GCaMP6f-expressing astrocytes over days. In a good window, the neuronal structures appeared crisp with clearly visible dendritic spines.

Two new spines appeared on day two of imaging. Only one spine persisted and was visible on day five. Calcium activity was studied in astrocytes expressing GCaMP6f at different time points.

A global event encompassing the entire cell was witnessed during a locomotion bout. The two critical steps of this protocol are that the bone needs to be adequately thinned prior to removal of the bone, and the proper placement of the cover glass over the opening. Imaging can be combined with behavioral analysis, either during imaging, or post-behavior, such as learning a new motor skill, to determine how neuronal and astrocyte structure and function is modulated by learning.