Installation of Optical Windows in the Brain of Transgenic Mice to Image Neural Activities
Trascrizione
Confirm a lack of response to toe pinch on an anesthetized 20 to 25 gram, 1 and 1/2 to 2 and 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-edged 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 2-by-3-millimeter cut in the scalp.
Using a cotton swab, push the skin aside to create an exposure area of greater than 3 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 3-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 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 2/3rd bone depth has been achieved, slowly and carefully thin the remaining one-third 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 stereomicroscope. 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 super glue 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. 7 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 brightfield illumination at the 4 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.
