This protocol allows us to identify axon guidance pathways active in the oculomotor nerve and to assess their roles at different points along the nerve trajectory in real time. This technique preserves the local environments through which axons travel and their final targets. The growing axons are not cut, so initial axon outgrowth, rather than regeneration, can be assessed.
This method provides insights into axon guidance in the ocular motor system but could be adapted to the study of axon guidance of other nerves. This technique takes practice to master, and working quickly is extremely important. When trying it for the first time, use just a few embryos, rather than a whole litter.
Before beginning the procedure, first confirm the pregnancy by ultrasound on embryonic day 10.5 from a timed mating. Harvest the embryos, spray the abdomen of the pregnant mouse with 70%ethanol, and use scissors to open the abdominal cavity to extract the uterus. Wash the uterus in a Petri dish of ice-cold HBSS before placing the organ in a second Petri dish of fresh ice-cold HBSS.
Under a dissecting scope, remove the embryos from the uterine horn and their individual amniotic sacs, placing each embryo on the underside of the lid of a 12-well plate, on ice, as they are harvested. Use filter paper to remove any liquid surrounding each embryo, without touching the embryos themselves, and submerge the embryos in liquid 4%low-melting agarose. Place the plate lid on ice to solidify the agarose.
When the agarose has hardened, flip over the embryos and cover the other side of each sample with additional agarose. When the second volume of agarose has solidified, use a fluorescence dissecting microscope to trim the agarose around each embryo so that each embryo will be oriented properly on the vibratome stage. The oculomotor nuclei and early axon outgrowths should be fluorescent.
Align each embryo so that the nucleus, outgrowing axons, and eye form a line, and use a razor blade to trim the agarose parallel to this line. Next, fill the vibratome chamber with ice-cold slice buffer, and superglue the first embryo to the vibratome stage so that the blade will be parallel with the oculomotor nucleus and eyes. When the superglue is dry, submerge the vibratome stage so that the embryo is oriented facing away from blade, and use a new vibratome blade to obtain 400-to 450-micrometer slices.
Use a sterile transfer pipette to transfer each slice into cold slicing buffer as it is acquired, and use the fluorescence dissecting microscope to select the slice containing the oculomotor nuclei and eyes. Using a sterile transfer pipette, transfer the slice onto a cell culture insert in a six-well plate containing 1.5 milliliters of culture medium per well, and place the plate in a 37-degree Celsius incubator. Remove the residual agarose from the vibratome stage, and superglue the next embryo onto the stage, continuing to collect slices until all of the embryos have been sectioned and plated.
Then add the appropriate concentration of the inhibitor or recombinant molecule of interest, diluted in an appropriate solvent, to the medium of each well. Create a dose-response curve. Image the slices every 30 minutes by phase contrast and fluorescence microscopy for up to 72 hours.
During normal in utero development, the first green fluorescent protein-positive oculomotor axons reach the orbit by embryonic day 11.5 before branching to their final targets, as observed in this representative slice culture. The orientation of the slice on the vibratome stage is crucial, as the slices are not interpretable if they're not oriented properly. For example, if the embryo is tilted to its side, only one oculomotor nucleus will be observed within the slice.
If the embedded embryo is tilted too far towards its back, the eyes will not be present within the slice, and instead the upper limb and hindbrain or spinal cord may be included. It is also important to take care that during the placement of the slice onto the culture membrane, that the tissue does not become folded. Take care when dissolving the inhibitors and growth factors in organic solvents.
For example, in this experiment, the slice died after the addition of ethanol to the medium. Remember to keep everything on ice and to minimize the time between the extraction of the embryos and placing the slices in the incubator. Using this technique, we have begun to identify additional axon guidance mechanisms at work in the ocular motor system.
Remember to use caution when working with razor blades and that some inhibitors may be hazardous and should be handled carefully according to their safety profiles.
