Isolation and Culture of Oculomotor, Trochlear, and Spinal Motor Neurons from a Mouse Embryo

Take a transgenic pregnant mouse and surgically remove the uterus with embryos containing fluorescently labeled motor neurons.

Transfer the uterus to a dish with an ice-cold buffer under a fluorescence microscope.

Use visible light to isolate the embryos.

Remove the face and tail of the embryos and orient them to a prone position.

Using the fluorescence signal, make an incision at the midbrain to expose the oculomotor and trochlear nuclei, which contain motor neurons.

Isolate the midbrain containing the nuclei.

Open the dorsal side of the hindbrain and spinal cord. Isolate the spinal cord, excise both ends, and collect the columns containing spinal motor neurons.

Treat the collected tissues with enzymes to dissociate the tissue matrix. Harvest the single cells and isolate fluorescently labeled neurons using FACS.

Add a culture medium to the isolated neurons, transfer the cells onto a culture substrate-coated microplate, and incubate, allowing the cells to adhere and grow.

Maintain the motor neurons in culture.

Begin by harvesting Islet-1 EGF-positive embryos from a pregnant mouse approximately 11.5 days post-fertilization. Spray the abdomen thoroughly with ethanol, and remove the uterus with sterile microdissection scissors and a thumb-dressing forceps. Briefly wash the uterus in sterile PBS. Then, transfer it to the dissection plate filled with pre-chilled sterile PBS.

Under the bright light of the microscope, carefully remove the embryos from the uterus using microdissection scissors, thumb-dressing forceps, and Dumont Number 5 tweezers. Then, use a sterile Moria mini-perforated Spoon to transfer each embryo to a separate well of a 24-well plate filled with pre-chilled Hibernate E low fluorescence medium supplemented with 1X V27.

While keeping the plate on ice, transfer one embryo to a sterile dissection plate, and cover it completely with ice-cold sterile Hank's Balanced Salt Solution or HBSS. Use tweezers to remove the face of the embryo and the tail without damaging the midbrain. Then, place the embryo prone with limbs straddled and tail pointing toward the front of the microscope.

Use tweezers to slit open the roof of the fourth ventricle in order to generate a small opening. Use this opening to hook tweezers into the space created between the fourth ventricle and its roof. Dissect along the dorsal surface of the embryo rostral to the cortex and lateral to the floor plate and motor column.

Then, open the dissected tissue in an open-book manner to reveal the GFP-positive CN3 and CN4 nuclei. Carefully separate the ventral midbrain from the embryo and remove meningeal tissue with tweezers and a microdissection knife.

Dissect the bilateral GFP-positive CN3 and CN4 nuclei away from the floor plate and other GFP-positive surrounding tissue, taking care to avoid touching or damaging the neurons. If collecting separate CN3 and CN4 nuclei, cut along the midbrain of these two nuclei, and use a P1000 pipette to collect the dissected ventral midbrain tissue with minimal HBSS.

Place it in a labeled 1.7-milliliter microcentrifuge tube filled with dissection medium. Store the tube on ice until dissociation. Continue pooling ventral midbrains from additional embryos in the same tube until the total number meets experimental requirements.

To dissect the ventral spinal cord, keep the embryo prone with the head facing the front of the microscope. Hold it with one pair of tweezers and insert the tip of the other pair into the unopened caudal part of the fourth ventricle. Open the rest of the hindbrain and spinal cord dorsally over the whole rostrocaudal extent of the embryo. Cut the dorsal tissue starting from the fourth ventricle and working toward the central canal of the caudal spinal cord, using the forceps as scissors.

Then, hold the embryo with one set of tweezers and pinch off the flap of the dorsal tissue on each side with the other pair. Remove the ventral spinal cord by using the microdissection knife to pierce directly below the GFP-positive SMN, lifting the ventral spinal cord with saw-like movements on both sides.

Cut the spinal cord transversely at the upper boundary of the lower limb, and remove the cervical lumbar portion. Cut transversely directly above C1 where the first GFP-positive anterior horn projects. Place the ventral spinal cord dorsal side up and hold it by pressing the GFP-negative tissue between the GFP-positive SMN columns with a pair of tweezers.

Remove the remaining attached mesenchyme, DRGs, and dorsal spinal cord by trimming both sides of the GFP-positive SMN column with the microdissection knife. Use a P1000 pipette to collect the dissected ventral spinal cord tissue with minimal HBSS, and place it in a labeled 1.7-milliliter microcentrifuge tube filled with dissection medium. Store it on ice until dissociation and continue pooling ventral spinal cords from additional embryos in the same tube.

Add the appropriate volume of papain solution to each of the microcentrifuge tubes with the dissected tissue samples. Incubate the tubes at 37 degrees Celsius for 30 minutes, agitating the tubes every 10 minutes by finger flicking.

After the incubation, gently triturate each suspension eight times with a P200 pipette. Centrifuge it at 300 times g for five minutes. Resuspend the cell pellets with the appropriate volume of albumin ovomucoid inhibitor solution by gently pipetting up and down. Repeat the centrifugation. Then, carefully remove the supernatant with a P1000 pipette. Resuspend the cells in the appropriate volume of dissection medium. Filter the suspensions through 70-micrometer cell strainers.

Next, use FACS sorting to isolate GFP-positive cells dissected from CN3, CN4, and SMN. Dilute the isolated cell suspensions with motor neuron culture medium pre-warmed to 37 degrees Celsius to the appropriate densities, and add 200 microliters of the suspension to a well of a PDL-laminin-coated 96-well plate. Culture the neurons in a 37 degrees Celsius and 5% carbon dioxide incubator, making sure to refresh the motor neuron culture medium every five days.