In Vivo Wide-Field and Two-Photon Calcium Imaging from a Mouse Using a Large Cranial Window

This protocol shows a creation of large cranial window to measure wide-field and single-cell reservation activities in the same mouse. Using our method, large and stable cranial window can be made with food wrap at low cost. This method is effective to study the neural and glial activities during behavior at macroscopic and microscopic levels.

It is recommended to start with a small cranial window. Then if it is succeeded, increase the size of the window. Begin by removing the extra cement over the skull with a dental drill.

Mark the area to be cut with a pen. Cut into the bone using a scalpel with a blunt tip to ensure that the tip does not penetrate the skull. Scrape the bone repeatedly with a scalpel to deepen the groove until the bone in the area to be trimmed moves when lightly touched.

Remove the incised bone with fine tweezers. Be cautious to not push the bone flap into the brain which may damage the brain. To remove the dura mater, cut the dura using a pulled glass pipette with a tapered tip of about 10 micrometers.

Use a U-shaped needle to expand this cut over the entire window. Under a stereo microscope set at 60 to 100x zoom, remove the severed dura mater with ultra fine tweezers. In case of bleeding, Rinse with aCSF or use a gelatin sponge to stop the bleeding.

Then, sterilize a large piece of PVDC wrap by autoclaving and with 70%ethanol. Under a stereo microscope, use tweezers and a scalpel to cut out a wrap of the required size. Place the wrap on the brain surface while leaving the aCSF on the surface.

Suck out the aCSF from the edge of the wrap, allowing the wrap to stick firmly to the brain surface. Trim the wrap with a scalpel and tweezers such that there is approximately a one millimeter margin between the edge of the cranial window and the wrap. Once the wrap is in place, glue the edge Of the wrap to the skull with a biological adhesive.

Allow the adhesive to dry for about 30 minutes. Using a dispenser with a mixing tip, apply a transparent silicone elastomer on top of the wrap. Place the cover glass of thickness 0.12 to 0.17 millimeters on top.

Seal the perimeter of the cover glass with waterproof film, superglue, or dental cement. First, mix fibroin and AAV solutions in a one to four ratio in a small sample tube. Drop an aliquot of the solution onto the plastic wrap for the cranial window and dry it for at least three hours.

Then attach the wrap to the brain surface as shown in the previous section. Check the condition of the mice and windows regularly, two to four weeks. By this time, the genetically encoded calcium indicators will be expressed sufficiently after creating the AAV-treated window.

Using this protocol, cranial windows could be maintained in eight of 10 mice for up to 10 weeks. A wide window is created with plastic wrap, transparent silicone, and cover glass in a transgenic mouse expressing membrane-anchored genetically encoded calcium indicator in astrocytes. Wide-field calcium imaging was performed through this window and fluorescence changes were observed when air puff stimulation was applied to the contralateral whiskers.

The time course of the fluorescence change in the somatosensory cortex showed cortical activity. Two-photon imaging allowed the observation of single cell fluorescence images specific to glial cells. Fluorescence changes induced by sensory stimulation were observed in individual cells from different cortical regions.

Wrap coded with AAV expressing genetically encoded calcium indicator;and fibroin was used for making the cranial window. Wide-field calcium imaging showed cortical activity propagating across the cortex induced by sensory stimulation two to four weeks after surgery. Since the window was large, different cortical areas of the same mouse were imaged and fluorescence changes induced by sensory stimulation were seen in individual cells.

It is important to avoid damage to the brain when making cranial window. One can perform behavior tasks on mice with a large cranial window. This will allow examining the relationship between neural activity and mouse behavior.

This method can be applied to a variety of neuroscience studies. For example, it can be used to observe cortical activity during decision-making tasks, motor running, and in mass models of brain injury and disease.