Primary Culture of Mouse Mesencephalic Neurons

Prepare two containers of crushed ice.

Clean the dissection surface with 70% alcohol. Place the dissection tools in a beakers filled with 70% alcohol and a Kimwipe (to protect the tips of the tools).

Prepare 1 petri dish with 2mL for 2 animals (n times) . Keep 37On ice.

Prepare 1 petri with 2mL for 5 animals (n times) to collect the tissue blocks. Identify the petri and keep 37On ice.

Prepare one 10 ml syringe per series of 5 animals (n times) with dissociation solution. Keep 37On ice.

Place the animals 37On ice. Wait 0h 2m 0s to 0h 3m 0s (until they are anesthetized). Put a maximum of 5 pups on ice at a time. Once the dissection begins, add the subsequent series of pups.

The first person removes the brains from the skulls once the animals are anesthetized (nonresponsive to manipulation and paw pressing). Wipe the skull and neck area with 70% alcohol and dry with a Kimwipe.

Hold the animal by the skin under the throat. Cut the skin around top of the skull (starting behind the ear). Take care not to damage the brain, keep the tips of the scissors facing upwards.

Remove the cut part of the skin and the skull with the curved forceps (pointing upwards).

Rinse the brain thoroughly with cold dissociation medium (2 ml per animal).

Using the curved forceps gently remove the brain and place it in a petri dish (start at the olfactory bulbs and finish by cutting the base of the brain stem).

The second person, using a binocular magnifying glass, dissects the isolated brains, one by one, preparing the slices and tissue blocks: Place the brain with the ventral region up and hold it gently with the thin forceps at the frontal lobe.

Using the scalpel blade, cut a thin coronal slice of the brain at the "midbrain flexure"; using the Willis circle as a reference (slice 1 mm thick).

Isolate the VTA and substantia nigra with a scalpel.

In the sterile hood, using a 10 ml sterile pipette, transfer the blocks of tissue obtained from a maximum of 5 brains into the 15 ml tube containing papain solution #1, taking care to transfer as little dissociation solution as possible with the tissue blocks, to reduce papain dilution. Incubate with stirring for 0h 20m 0s at 37°C. Quickly continue dissecting the brains of the other mice, transferring the resulting tissue blocks into the tubes containing papain solution #2, #3 and #4 (as needed) and incubate each with agitation 0h 20m 0s at 37°C.

Prior to starting, flame polish the tip of a 5 ml glass pipette to a diameter of 1.5 mm and a second to a smaller diameter (~0.5 mm, see annex 1).

After 0h 20m 0s of agitation, replace the papain solution in tube #1 with 2mL. Repeat this rinse a second time.

Rinse with 2mL, remove and add 1mL.

Gently triturate 20 times with the 1.5 mm pipette and 40 times with the 0.5 mm pipette ( use a rubber bulb ).

Repeat steps 17 to 19 with papain tube #2, #3 and #4 (as needed).

Gently transfer the dissociated cells, from the different tubes, on top of the centrifugation solution (no more than 2 ml of suspended cells per 5 ml centrifugation tube).

Centrifuge for 0h 2m 0s at position 3 (1150rpm,0h 0m 0s), then 0h 3m 0s at position 4 (1400rpm,0h 0m 0s) on a clinical IEC centrifuge (the centrifuge tubes must be balanced, calibrate the centrifuge with a 15 ml tube filled with water if needed).

Remove the supernatant and resuspend the cells in a 500µL. Mix 10µL with 10µL, take 10µL and count the cell density using a hemacytometer. Add to the cell suspension the required volume of the trituration medium (supplemented with Neurocell+ if necessary) to achieve the desired concentration. For the volume to add according to the number of cells and the desired concentration, see the calculation table. Combine the tubes of the same cell type in a single tube.

Dilution of the Cell Suspension

Apply 65µL to the coverslips covered with astrocyte monolayer. Remove 5 petri dishes at a time. Dry the bottom of the coverslips by placing them carefully one by one on a sterile Whatman filter paper. Place the coverslips in pre-sectioned (see annex 2) sterile dry petri dishes, and add the cell suspension at the desired concentrations as quickly as possible to avoid over drying the astrocyte monolayer. Do this step in pairs, the first person removes the coverslips from the old petri dishes, drying them on the Whatman filter paper, the second one recovers them and places them in a new petri, adding immediately 65µL. Place the petri in the incubator.

After 3h 0m 0s, add 2.5mL (Mix Neurocell+/EMEM+ for dopamine neurons, Neurocell+ only if striatal cells) to each petri dish and put back all the petris in the incubator.

After 24h 0m 0s, add 10micromolar (µM) (from 2 mM stock; 12.5 µl/2.5 ml) to inhibit glial proliferation.

If necessary , following 7 days of culture, add 0.5millimolar (mM) (from 125 mM stock, 10 µl/2.5 ml) to prevent the toxicity of glutamate release.

If necessary , add 500µL (Mix Neurocell+/EMEM+) every 5 days to all the petri dishes to compensate evaporation and to feed the cells.