This method enables the recording and quantification of neural activity across mouse bilateral cortical regions for a prolonged period in vivo. The two photon technique has the advantage of detecting activity simultaneously in large or dispersed neuronal population over an extended period. The implications of this technique extended toward the investigation of activity patterns in defined neuronal populations in different regions of the living brain.
12 hours before beginning the procedure, warm the optical windows at 50 degrees Celsius in 70%ethanol. An optical window consists of a round five-millimeter diameter top cover glass and a bottom portion containing a round three-millimeter diameter cover glass. At the end of the warming incubation, confirm a lack of response to toe pinch on an anesthetized 20 to 25-gram 1 1/2 to 2 1/2 month old Thy1 GCaMP6s mouse, and apply ointment to the animal's eyes.
Place the mouse on a heating pad covered with a sterile drape and use a head-holding adapter for mice to stabilize the head. Shave most of the scalp with a double-edge razor, and clean the exposed skin with sequential sterile alcohol preparation pads and povidone-iodine solution scrubs. Then use a pair of scissors to make a rectangular two by three millimeter cut in the scalp on the right side of the skull.
Using a cotton swab, push the skin aside to create an exposure area of greater than three millimeters in diameter, and use a blunt microsurgical blade to gently remove the connective tissue attached to the skull. With a dental drill, gently mark a three millimeter diameter circle around the S1 area. After making a similar circle on the left side of the skull, apply a thin layer of cyanoacrylate superglue to both sides of the bone to provide a base for a dental cement application.
Under a dissecting microscope, use a high-speed micro drill to thin down a circular groove in the right side of the skull around the S1 area to create a smooth edge, aspirating the bone debris with a vacuum as necessary. After an approximately 2/3 bone depth has been achieved, slowly and carefully thin the remaining 1/3 of bone until a circular bone flap has been completely freed from the surrounding skull. Use a pair of 5/45 forceps to slowly and carefully remove the circular piece of bone to expose the dura, taking care to avoid damaging the pial vessels.
After removing a bone flap from the left side of the skull in the same manner, rinse the optical window for the right side of the skull with sterile saline, and check for imperfections under a stereo microscope. Install the optical window over the craniotomy region with the top portion of the window resting on the skull and the bottom portion within the craniotomized opening, resting on the dura, in the presence of cerebral spinal fluid. Use the cyanoacrylate superglue to seal the top portion of the optical window edge to the skull.
When the glue has dried, apply black dental cement to the edge of the glass, the rest of the right side of the exposed skull, and the wound margins to block the light. Then install the left optical window and allow the animal to recover with monitoring until full recumbency. Seven to 10 days after the procedure, place the anesthetized animal into the head-holding adapter on a heating pad under a two-photon microscope, taking care that the optical window and the right side of the skull are oriented perpendicular to the optical axis of the microscope.
Acquire an initial reference map image of the cranial window using bright-field illumination at the four times magnification. After acquiring multiple images of the same region of interest, image the calcium dynamics within the S1 area in the left hemisphere and calculate the changes in the fluorescence from each region of interest on both sides of the skull. In these representative images, dendrites and a blood vessel within an EGFP-expressing mouse were visualized at different depths in layer two of the S1 region one day after installation of the optical window.
The Z-projection images were captured at days one and seven after the optical window implantation, illustrating a remarkably stable number and location of dendritic branches and spines over the experimental period. Measurement of the intracellular calcium transient in Thy1 GCaMP6s transgenic mice allows the quantification of spontaneous calcium activity and populations of layer five pyramidal neurons located as deep as greater than 500 micrometers below the pia as well as calculation of the average number of spontaneous responses over time. A researcher trained in both cranial window preparation and in vivo two-photon calcium imaging techniques should be able to obtain recordings of 100 to 200 neurons on both sides of the skull in one to two animals per day.
With an open optical window on each side of the skull, the method enables the recording of the neural activity across symmetric mammalian brain regions for prolonged and stable calcium imaging in vivo. This method can also be used to study cortical activity and plasticity after unilateral injury to the peripheral or the central nervous system. After watching this video, you should have a good understanding of how to install bilateral cranial windows for measuring calcium dynamics using two-photon microscope in Thy1 GCaMP6s transgenic mice.
