An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

The overall goal of this procedure is to visualize and quantify the engulfment of presynaptic inputs by microglia. This is accomplished by first injecting the fluorescent anterior grade tracers into the eyes to label retinal ganglion cell presynaptic inputs in the lateral genant nucleus. The second step is to dissect, fix, and equilibrate the brain.

Next, the brain is sectioned. The final step is to mount the brain sections on a cover lip for imaging and analysis. Ultimately, confocal microscopy is used to assess the engulfment of presynaptic inputs by microglia.

This method can help answer key questions in the neuroscience field, such as how phagocytic soils contribute to synaptic circuit plasticity and remodeling. Though this method can provide insight into synaptic circuit remodeling in the healthy brain, it can also be applied to other systems such as synaptic circuit remodeling during nervous system disease. Demonstrating the procedure will be Chris Heller, a technician, and Emily Laman, a grad student from the Stevens Laboratory.

Begin this procedure by anesthetizing a mouse with 4%ISO fluorine in a plexiglass induction chamber. After one to three minutes, ensure an appropriate level of anesthesia is achieved by pinching the tail. Then place the mouse on its side under the stereo microscope and place the nose cone, which delivers three to 4%ISO fluorine over the snout.

To expose the sclera, use a pair of small spring scissors to open the eyelid and pull back the skin. For neonates, sometimes another perpendicular cut at the corner of the eye is necessary. Be careful when cutting the corner of the eyelid as there is a blood vessel.

Use a sterile 30.5 gauge needle to puncture a small hole at the side of the eye where the sclera begins. Take care to avoid damaging the lens by inserting the needle just far enough so that the bevel goes into the eye. Allow the vitreous to flow out of the hole and use a sterile cut and tipped applicator to absorb the liquid.

Once the vitreous has stopped flowing out of the hole slowly insert a blunt ended needle attached to a Hamilton syringe that has been preloaded with anterior grade tracing. Dye into the hole, slowly inject the dye into the eye. Typically, cholera toxin beta subunit conjugated to Alexa 5 94, 6 47 or 4 88 is used to anterior grade.

Late trace R GC inputs Leave the needle in the hole for a few seconds and then remove it slowly. Use a cotton tipped applicator to absorb excess fluid and prevent the dye from leaking out. Then apply a small amount of antibiotic ointment to the eye.

If the eye was surgically opened. Gently reposition the eyelids together following injection. Leave the mouse under a heat lamp or on a heat pad until it begins to recover from the anesthesia.

Return the mouse to a clean home cage and monitor it to make sure it is fully awake before returning it to the colony. About 24 hours after the injection, sacrifice the mouse and dissect the brain. Fix the brain in a Falcon tube filled with 4%PFA overnight at four degrees Celsius the next day.

Rinse the brain three times in PBS by first pouring the brain and PFA into an empty whey boat Then uses spatula to transfer the brain to another whey boat filled with PBS. Wash the brain two more times in PBS before transferring it to a Falcon tube filled with 30%sucrose solution. Leave it in sucrose at four degrees Celsius until it sinks to the bottom of the tube.

Afterward, remove any parts of the brain that are not needed with a razor blade. Freeze the brain on a piece of aluminum foil over dry ice. In the meantime, freeze the microtome stage and fill each well of a 24 well plate with half a milliliter of 0.1 molar PP to mount the frozen brain on the freezing stage.

Apply a small amount of OCT to the stage. Once the OCT begins to freeze, lay the brain in it. The side of the brain that will be cut should be facing up.

Next, cover the brain and OCT with very finely crushed dry ice. Leave the dry ice on the brain for about 30 seconds. Then use a large paint brush to remove the dry ice.

Begin sectioning the slices at 40 micron thickness. Then use a small moist paintbrush to remove the sections containing the region of interest from the blade and transfer them to the 24 well plate containing 0.1 molar pb. Once the sections have been collected, visualize the anterior grade labeling under a fluorescent dissecting microscope and choose sections that contain the region of interest to mount the sections on a slide.

First, apply a small pool of 0.1 molar PB to a charged microscope. Slide next, transfer the tissue sections to the pool of pb. Then use the paintbrush to orient and spread the tissues.

Use a Kim wipe to wick off XSPB and take care to avoid wicking off the section. Allow them to air dry completely. Then apply a small drop of mounting medium to each section and mount a cover slip on top at the end.

Seal the edges of the slide with nail polish. Store the slide at minus 20 degrees Celsius until the imaging session shown. Here is a schematic of anterior grade tracing strategy.

The left and right eye RGC inputs are traced with CTB 6 47 and CTB 5 94 respectively. The microglia mediated engulfment of inputs is subsequently assessed. Here is a representative, low magnification image of postnatal day five Mouse DLGN following intergrade tracing of left and right eye inputs.

This is a microglia sampled from the border region of left and right eye inputs. All CTB fluorescence outside the microglial volume has been subtracted revealing the RGC inputs that have been engulfed and the surface rendering of microglia and engulfed. RGC inputs is shown here.

Here is the representative surface rendered microglia from P five, P nine and P 30 mouse. DLGN Engulfment of RGC inputs is significantly increased during peak pruning in the DLGN versus older ages. Microglia from mice deficient in complement receptor three engulf significantly fewer RGC inputs as compared to wild type litter mates Once mastered.

This technique can be done in 72 hours if it is performed properly. After watching this video, you should have a good understanding of how to intergrade label neurons and assess microglia synapse interactions.