To fully characterize a microglial phenotype, it's important to address both basal and directional motility. This protocol can be used to test the directional motility of microglia by two-photon imaging in mice brain slices, using customized 3D print interface and perfusion chambers. Demonstrating the procedure will be Fanny Etienne, a Ph.D.student, and Vincenzo Mastriolia, a post-doctoral researcher, both from our laboratories.
At least 30 minutes before the dissection, start bubbling 70 milliliters of choline artificial cerebrospinal fluid, or choline aCSF, on ice. Add 150 milliliters of choline aCSF at 32 degrees Celsius, with carbogen. Place a 200 milliliter crystallizing dish with a bar magnet into a sealed food box and add 200 milliliters of aCSF to the dish.
Place a 3D printed interface slice holder on top of the dish and remove excess fluid from the crystallizing dish, until only a thin film of solution covering the mesh of the interface slide holder remains. Add a few millimeters of aCSF at the bottom of the food box and start bubbling the fluid with carbogen. Then, close the sealed box while maintaining constant carbogenation to create a humidified, 95%oxygen, 5%carbon dioxide-rich interface chamber.
After harvesting, place the brain onto a piece of aCSF-soaked filter paper and dissect out the region of interest, according to the preferred angle of slicing. For coronal slices, position and glue the caudal face of the brain onto a 10 centimeter Petri dish attached to the cutting block of a vibrating slicer, and position the block in the reservoir chamber of the vibrating slicer within a large chamber filled with ice. Fill the dish with ice-cold choline aCSF and use the slicer to obtain 300-micrometer thick brain slices, using a four millimeter diameter disposable transfer pipette after every pass of the blade to collect each slice.
Let each slice recover in the 32 degree Celsius choline aCSF for about 10 minutes after it is obtained, before transferring the slices onto pieces of lens cleaning paper, topped with a drop of choline aCSF. Then aspirate the excess choline aCSF and use a spatula to transfer the slices onto the mesh of the interface chamber, allowing the slices to recover in this environment for at least 30 minutes. 30 minutes before starting the recording, connect the peristaltic pump to a customized recording chamber with top and bottom perfusion for optimizing the oxygenation and viability of the tissue slices, and clean the entire perfusion system with 50 milliliters of ultrapure water.
At the end of the cleaning cycle, start the perfusion of the recording chamber with 50 milliliters of aCSF in a glass beaker under constant carbogenation, and use a disposable, wide-mouth transfer pipette to transfer the first slice to be imaged to the beaker to remove the lens paper. When the section has sunk to the bottom of the beaker, transfer the section to the recording chamber and position the slice holder onto the slice to minimize movement induced by the perfusion flow. Use bright-field illumination to target the brain region of interest under a five to 10x magnification.
Then, use a 25x objective with a 0.35x water immersion lens to adjust the position of the viewing field. Use fluorescence illumination to locate the fluorescent microglial cells and backfill the pipette with 10 microliters of aCSF containing the compound of interest at its final concentration. Point the tip downward with gentle shaking to remove any air bubbles trapped in the tip and mount the filled pipette into a pipette holder connected to a five milliliter syringe with the plunger positioned at the five milliliter position and mounted onto a three-axis micromanipulator.
Lower the pipette gently toward the slice, controlling and adjusting the objective at the same time, until the pipette tip lightly touches the surface of the slice. Now tune the laser and switch the microscope to the multiphoton mode. Make sure that chamber is screened from any light source and switch on the non-descanned detectors.
Set the gain and use a lookup table with a color-coded upper limit to avoid saturating the pixels in the image. Then, start recording for a total duration of at least 30 minutes, slowly depressing the syringe plunger from the five to one milliliter position, after five minutes, over a period of five seconds, to apply the compound to the section. For image analysis, first perform z-projection and drift correction with ImageJ on the file of interest.
Then, open the modified file in Icy and draw a circular, 35-micrometer diameter region of interest, centered over the injection site. Run the movie again with the ROI, to ensure that it is well-positioned. Then, use the region of interest intensity evolution plugin to measure the mean intensity over time in the region of interest, and save the results as a xls file.
This protocol allows the responses induced by different compounds, like ATP or serotonin, to be measured. Although the injection of ATP elicits an increase of fluorescence, after most ATP injections, there is an immediate, slight decrease in fluorescence due to tissue distortion that comes back after a few minutes. The size of the region of interest also impacts the quantification, as increasing the diameter reduces the variability among the experiments, but decreases the accuracy and magnitude of the detected response.
Assessment of the microglial response at a specific time point can be useful for the statistical comparison of different compounds, or to test the effects of specific antagonists added to the perfusion solution. To quantify the motility in three dimensions, know that you may have to change the z-step interval and the sampling rate of the acquisition to fit within the analysis requirements.
