Our protocol enable the simultaneous imaging of microglia dynamics and neuronal activity in awake mice. It can be widely applied to investigate the surveillance behavior of microglia or the interaction with neurons. The advantage of this method is that motion artifact do not easily contaminate imaging data because of robust head fixation.
Also, restoration after imaging at the high frame rate allows us to eliminate motion artifact from the data. In this method, the AAV injection and the surgery of cranial window implantation are technically demanding. Failure is common at the beginning, so please don't get frustrated and practice more.
To prepare the injection apparatus, place a glass pipette connected to a 26-gauge Hamilton syringe through a tube onto a pipette holder of a stereotaxic instrument. Then, tilt the pipette holder 60 degrees anteriorly from the vertical axis. Fill the glass pipette, the syringe, and the connecting tube with liquid paraffin and place the syringe on a microinjector.
Administer analgesia to the anesthetized mouse and attach the auxiliary ear bars. Then, fix the animal on the stereotaxic instrument with its dorsal side up. After removing hair from the surgical site and disinfecting the surgical area, make a two-centimeter long incision on the scalp along the midline, ensuring a good exposure of the skull above the right primary visual cortex.
Use forceps to remove the periosteum on the exposed skull. Drill the skull on the stereotaxic coordinates three millimeters lateral to the midline and 5 milliliters anterior to the lambda line to create a small hole with a diameter of approximately 0.5 millimeters. Then, place a piece of transparent film approximately two centimeters by two centimeters on the exposed skull of the mouse.
Expel one microliter of AAV solution droplet on the film using a pipetter. Advance the syringe. And place a glass pipette tip into the droplet of AAV solution on the film and gently pull the syringe to aspirate the AAV solution.
Next, insert the glass pipette to a depth of 500 micrometers from the brain surface through the hole created in the skull. Then, inject 0.5 microliters of AAV solution using the microinjector at an injection volume flow rate of two microliters per hour. Withdraw the needle and rinse the brain surface with saline.
Create a circular groove along the mark on the skull by drilling. Clean the debris to ensure visibility of the skull and apply saline to prevent heating during drilling. Gently press the central skull with forceps.
The drilling depth is sufficient if it moves vertically with little resistance. When the groove reaches enough depth, insert the tip of the forceps into the bottom of the central skull fragment. Gently lift and remove it to expose the brain surface.
Using a 27-gauge needle, prick and tear the dura at the edge of the exposed brain surface. Insert the tip of the forceps through the hole made at the edge of the dura and peel it off to expose the brain surface. After dispersing hemostatic fibers one by one in saline, place the hemostatic fibers along the margins of the hole in which the transected dura is present.
Place the cranial window on the exposed brain surface and then adhere it to the skull with instant glue while gently pressing the window. Afterward, apply instant glue to the whole exposed skull. Then, carefully attach a head plate onto the skull to locate the cranial window at the center of the square hole of the head plate.
Once the glue has sufficiently hardened, apply dental cement to the exposed skull to reinforce the attachment between the head and the head blade. To install the custom made shading device and an LCD monitor for visual stimulation, first position the lens to focus on the brain surface and then set this lens position as the original Z position. Keep the XY coordinates constant and elevate the objective lens.
Remove the mouse and stereotaxic instrument from the objective lens. Attach a shading device on the top of the head plate using silicone, ensuring that the space between the head plate and the shading device is well-sealed. Fill the shading device with distilled water.
Then, fix the mouse with the stereotaxic frame under the objective lens. Carefully reset the focal plane at the brain surface, checking the depth of the objective lens. Cover the objective lens with black aluminum foil to avoid light contamination from the LCD monitor.
Set a 10-inch LCD monitor at 12.5 centimeters in front of the eyes of the mouse to present visual stimuli. Configure the fluorescent submission collection filters for EGFP and R-CaMP and the excitation wavelength to 1, 000 nanometers. Acquire images with a spatial resolution of 0.25 microns per pixel.
Find the imaging region where R-CaMP-positive neurons and EGFP-positive microglia can be simultaneously imaged. In layer 2, 3. Acquire images at the frame rate of 30 hertz.
Simultaneously with the image acquisition, present drifting grating visual stimuli to the mouse in 12 directions at six orientations, from zero to 150 degrees in 30-degree steps. After the image acquisition, remove the mouse from the microscope stage. Detach the shading device and the stereotaxic instrument from the mouse, and return the mouse to its home cage.
Using this protocol, AAV injection and cranial window implantation were performed in the primary visual cortex of an eight-week-old transgenic mouse, followed by two photon imaging of R-CaMP-based neural activity and microglial dynamics in layer 2, 3. Grating visual stimuli were presented to the mouse and the visual responses in adendritic spine were analyzed using calcium traces. Microglial processes showed fast dynamics and changed their morphology within 10 seconds.
Using two-photon imaging in layer 2, 3 of the primary visual cortex in a 12-week-old transgenic mouse, R-CaMP was observed in neurons, seen here in magenta. An EGFP was observed in microglia, seen in green. Individual signals were also observed for EGFP and R-CaMP.
Successful AAV injection is critical for this method. The main reasons for failed AAV injection are the clock glass pipettes and the tissue damage. The surveillance behavior on microglia and the SNAP's microglia interaction have been identified to be prevalent in various pathogenic mechanisms, like Alzheimer's disease.
Our method is beneficial to the research focused on this field.
