TITLE: Primary Culture of Mouse Mesencephalic Neurons
AUTHORS: Nicolas Giguère

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the procedure required for the dissection and collection of primary mouse culture mesencephalic neurons.</div></div>

[STEPS]
?. [Dissection (preferably in pairs)]
Prepare two containers of crushed ice.
?. [Dissection (preferably in pairs)]
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).
?. [Dissection (preferably in pairs)]
Prepare 1 petri dish with  for 2 animals (n times). Keep .
[dissociation medium]
on ice
?. [Dissection (preferably in pairs)]
Prepare 1 petri with  for 5 animals (n times) to collect the tissue blocks. Identify the petri and keep .
[dissociation medium]
on ice
?. [Dissection (preferably in pairs)]
Prepare one 10 ml syringe per series of 5 animals (n times) with dissociation solution. Keep .
on ice
?. [Dissection (preferably in pairs)]
Place the animals . Wait  to  (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.
on ice
?. [Dissection (preferably in pairs)]
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.
?. [Dissection (preferably in pairs)]
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.
?. [Dissection (preferably in pairs)]
Remove the cut part of the skin and the skull with the curved forceps (pointing upwards).
?. [Dissection (preferably in pairs)]
Rinse the brain thoroughly with cold dissociation medium (2 ml per animal).
?. [Dissection (preferably in pairs)]
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).
?. [Dissection (preferably in pairs)]
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.
?. [Dissection (preferably in pairs)]
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).
?. [Dissection (preferably in pairs)]
Isolate the VTA and substantia nigra with a scalpel.
?. [Dissection (preferably in pairs)]
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  at . 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  at .
37 °C
37 °C
Important: Keep the tissue blocks and solutions on ice for as long possible and perform the entire dissection, from the first skin perforation to the last tissue block being placed in papain as quickly as your dexterity allows. The health of cells and their survival depends entirely on this. The procedure for brain extraction and block cutting should be 1 minute or less, per animal.
?. [Dissociation of Cells]
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).
?. [Dissociation of Cells]
After  of agitation, replace the papain solution in tube #1 with . Repeat this rinse a second time.
[dissociation solution]
?. [Dissociation of Cells]
Rinse with , remove and add .
[trituration solution]
[fresh trituration solution]
?. [Dissociation of Cells]
Gently triturate 20 times with the 1.5 mm pipette and 40 times with the 0.5 mm pipette (use a rubber bulb).
Trituration is not complete if you still see pieces of tissue (cells suspension should be homogeneous without any piece of tissue). If you ask yourself why this might happen, the possible problems are: the size of the tissue blocks, inactivated papain, excessively long dissection time, etc. However, the following can be attempted: let the undissociated pieces fall to the bottom of the tube, transfer the cells in suspension into another sterile 15 ml tube, add 2 ml of trituration solution to the tube of undissociated pieces, and repeat the trituration a second time and finally pair the 2 tubes.
?. [Dissociation of Cells]
Repeat steps 17 to 19 with papain tube #2, #3 and #4 (as needed).
?. [Dissociation of Cells]
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).
?. [Dissociation of Cells]
Centrifuge for  at position 3 (), then  at position 4 () on a clinical IEC centrifuge (the centrifuge tubes must be balanced, calibrate the centrifuge with a 15 ml tube filled with water if needed).
Centrifuge: 1150 33
Centrifuge: 1400 33
?. [Dissociation of Cells]
Remove the supernatant and resuspend the cells in a . Mix  with , take  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.
[trituration medium]
[cell suspension]
[Trypan blue (Gibco, 15250061)]
[of the mixture]
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?. [Dissociation of Cells]
Apply  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 . Place the petri in the incubator.
[cell suspension]
[cell suspension]
Do not allow the coverslips to touch the edges of the petri dish. Handle the petri dishes with great care – note that the droplet will behave differently if the surface is already wet or not. When adding neurons to a coverslip with an astrocyte monolayer (that is moist) it is more likely to spread to the edges because of the effect of surface tension on the 65µl cell suspension droplet is reduced.
?. [Dissociation of Cells]
After , add  (Mix Neurocell+/EMEM+ for dopamine neurons, Neurocell+ only if striatal cells) to each petri dish and put back all the petris in the incubator.
[culture medium]
?. [Dissociation of Cells]
After , add  (from 2 mM stock; 12.5 µl/2.5 ml) to inhibit glial proliferation.
[FUDR]
?. [Dissociation of Cells]
If necessary, following 7 days of culture, add  (from 125 mM stock, 10 µl/2.5 ml) to prevent the toxicity of glutamate release.
[kynurenic acid]
?. [Dissociation of Cells]
If necessary, add  (Mix Neurocell+/EMEM+) every 5 days to all the petri dishes to compensate evaporation and to feed the cells.
[culture medium]