Primary Culture of Mouse Dopaminergic Neurons

The overall goal of this procedure is to show how to generate a primary culture of dopaminergic neurons from embryonic mouse brains. This is accomplished by first collecting mouse E 13.5 embryos. The second step is to dissect the embryonic mouse brains and to isolate the mesencephalon.

Next, an enzymatic and a mechanical dissociation are applied to the collected mesencephalon. The final step is the plating of dissociated neuronal cells. Ultimately, immunofluorescent staining is used to show the presence of dopaminergic neurons in the culture.

This method can help answer key questions in the field of neurological and psychiatric disorders such as schizophrenia, attention deficit hyperactivity disorder, and Parkinson's disease. Visual demonstration of this method is critical as the dissection and cell dissociation steps are difficult to learn because precise gesture and anatomical identification of the structure are required, demonstrating the procedure will be Florence given an engineer from my group To begin this procedure. After sacrificing an E 13.5 pregnant mouse, clean its abdomen with 70%ethanol.

Next, open the abdomen wall and collect the uterine horns. Dissect each embryo out from the uterine horns and remove the amniotic membranes. Then transfer the embryos to a 100 millimeter Petri dish containing sterile P-B-S-G-A-B.

Wash them by transferring in three successive identical baths. After that, transfer the embryos to a 60 millimeter sterile Petri dish For dissection. Place the Petri dish under the stereo microscope.

Next, excise the brain using Vanna Scissors. Carefully remove and discard the four and hind brain regions. Make a cut close to the thalamic region at the rostral side, and another cut in the ssus region at the coddle side.

Then remove the superior colus. Once the ventral midbrain is isolated, carefully remove meninges Using ultra fine forceps, collect the dissected segments without the meninges in a 13 milliliter sterile tube filled with P-B-S-G-A-B. Clean the tube containing the mesencephalon thoroughly with 70%ethanol.

Then place it under the hood in this step. Wash the mesencephalon three times with sterile P-B-S-G-A-B. Allow the brain fragments to settle between each wash and avoid disrupting the tissue after the last wash.

Carefully remove as much solution as possible and incubate the brain segments in three milliliters of trypsin EDTA for 15 minutes at 37 degrees Celsius in a carbon dioxide incubator. In the meantime, fire polish PEs your pipettes to maximize the survival of neurons as the end of a non polished glass Pipette is sharp and can damage the cells during the dissociation steps slightly. Reduce the extremity diameter of the PE pipette while fire polishing it.

Now carefully remove as much trypsin EDTA solution as possible. Then add 10 milliliters of culture medium, 10%IFBS, and wash the brain segments three times with it. Using the fire polished PEs pipette, begin mesencephalon dissociation in six milliliters of culture.

Medium, 10%IFBS trier rate 10 times and try to avoid air bubbles. Then allow the chunks to settle afterward. Collect the medium in a new sterile 13 milliliter tube.

Add six milliliters of culture, medium and tri rate 10 times more. Allow the chunks to settle and collect the medium containing the dissociated cells in the same 13 milliliter tube. Then centrifuge the cells at 160 Gs for five minutes.

After that, discard the supernatant and resus. Suspend the cells in culture medium supplemented with 10%hormone. Mix count cells in suspension in a mase cell, and adjust the volume of medium to a concentration of 600, 000 cells per milliliter.

In general, around 1, 700, 000 cells can be obtained from one mouse mesencephalon. Now remove FBS containing coating medium from a 24 well plate. Add one milliliter of cell suspension to each.

Well then incubate the plate at 37 degrees Celsius and 5%carbon dioxide 95%air dopaminergic neurons are mature after around five to seven days in vitro and the cells can be kept in culture up to 15 days without medium replacement. The day before the dissection, add 10 milliliters of one molar HCL into a Petri dish. Place 12 to 15 glass cover slips on the liquid surface and wait for 15 minutes.

Then press the cover slips into the liquid and wait for another 15 minutes. Afterward, remove the HCL and wash the cover Slips three times with water, followed by one quick wash with pure ethanol. Then add pure ethanol to the dish and wait for 30 minutes under the hood.

Remove the cleaned cover slips from ethanol. Subsequently transfer the cover slips to a 12 well cell culture plate. Wash the cover slips twice with sterile water, followed by washing them once with sterile PBS.

Then remove the PBS and add two XPLO at 500 microliters per well incubate for four hours at 37 degrees Celsius in the carbon dioxide incubator After four hours. Remove the PLO solution and wash three times with sterile PBS. After that, remove the PBS and add 500 microliters of 20%IFBS laminin at one microgram per milliliter to each well incubate overnight at 30 degrees Celsius in the carbon dioxide incubator.

For immunofluorescence. Remove the coating medium from the 12 well plate. Add two milliliters of cells at 900, 000 cells per well and grow them in the incubator At 37 degrees Celsius.

The cells can be kept in culture up to 15 days without medium replacement.Shown. Here are the phase contrast images of the culture at different stages of development. This figure shows the tyrosine hydroxylase staining of dopaminergic neurons at DIV eight D neurons were detected using anti TH antibody.

The number of TH positive neurons in mesencephalon culture is a function of the age of the culture. Here are the representative neurons and astrocytes in the culture. At DIV four, dopaminergic neurons were detected using anti DAT or anti TH antibody and the GABAergic neurons.

Serotonergic neurons and astrocytes were detected using anti GAD 67 anti serotonin and anti GFAP antibodies, respectively. The neuronal cells were stained with an anti map two antibody. Here is the representative staining of several cell populations of the mesencephalon culture.

A dopaminergic neuron was detected by DAT staining at DIV nine and these GABAergic neurons were visualized by GAD 67 staining at DIV nine. The serotonergic neuron was stained with an anti serotonin antibody at DI nine. The neurons were identified by map to staining After its development in the early eighties.

This technique paved the way for researchers in the field of neuroscience to explore dopaminergic cell development in primary culture and can be employed to investigate molecular mechanisms underlying physiological and pathological states of dopaminergic neurons. The lack of video presenting the delicate dissection may have restrained researcher to develop this mouse culture model. After watching this video, you should have a good understanding of how to precisely isolate functional from the embryonic mouse brain and conduct dedicate dissociation steps in order to obtain a nice primary culture of mouse dopaminergics.