Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

The goal of this procedure is to efficiently isolate and culture primary neurons from rodent ventral midbrain. This is accomplished by first dissecting the midbrain of rodent embryos. The second step is to carefully isolate the ventral midbrain.

Next, the neuronal tissue has to be dissociated to yield a single cell suspension. The final step is to plate the primary neurons in a small volume on cover slips before transferring the attached cells into a 24 well plate late. Ultimately, th antibody and immunofluorescence microscopy is used to identify the dopaminergic neurons within the culture.

The dopaminergic neurons in the midbrain are involved in several functions, including the control of movement and emotion in cognition and the reward secretory of the brain. They're also implicated in neurological and neuropsychiatric conditions, including Parkinson's disease, addiction, and schizophrenia. The method of repairing ventral midbrain.

Neuronal cultures can be used for studying the physiology and cellular properties of the dopaminergic neurons, as well as for understanding the cause of their degeneration. During the course of disorders such as Parkinson's disease. First, prepare the cover slips.

Boil them in 70%ethanol for at least 30 minutes to remove any traces of grease. Then autoclave the cover slips. Transfer the cover slips to 24 well plates and add half a milliliter of polyol all athene to each.

Well let the plates incubate for an hour at room temperature. Take note that the shelf life of working polyol or Athene solution is one month. Then wash the cover slips three times with half a milliliter of water per wash.

Without all three washes, the cultured cells would not survive 24 hours. Next, repair the laminin solution. Be sure to first thaw it slowly on ice.

Then dissolve the laminin in cold D-M-E-M-F 12, and use it immediately. Now at half a milliliter of freshly made working laminin solution to each well and let the plates incubate overnight at 37 degrees Celsius. Lastly, when preparing the glass pipette, I polish their tips to half their normal size.

Make an abdominal incision cision to expose the uterine sacs and using forceps, cut free the uterus. Transfer the uterus to ice cold HBSS in a 100 millimeter dish, and begin removing the embryos with forceps. Transfer the isolated embryos to a new dish of cold HBSS.

Now under a dissection microscope, prepare to access this location of the ventral midbrain under 10 x magnification. Dissect the meas and cephalic arch by cutting the brain at the isthmus and the meas and cephalic di cephalic boundary region Using bio scissors or forceps. After taking out the entire midbrain, make a cut in the medial dorsal midbrain.

Using forceps, remove the meninges. This step is critical to neuronal culturing success. Non neuronal cells will die under culture and conditions and adversely signal neighboring neurons.

Now flatten the midbrain tissue into a butterfly shape. Cut off about half of each wing with a sterile blade leaving the ventral midbrain. Transfer the ventral midbrain into ice cold HBSS in a 15 milliliter conical tube.

Then proceed to dissect the next embryo process as many embryos as possible within an hour. Embryos kept on ice for more than an hour, have poor cell viability. To ensure high viability of the dopaminergic neurons, the time from dissection until plating should not exceed one hour.

In addition, removing the meninges is crucial for the survival of the neurons Under a hood. Remove the HBSS from the collection of ventral midbrain. Then add a milliliter of warm 0.05%tripsin A DTA, enough solution for 12 pieces of tissue.

Incubate the tissue at 37 degrees Celsius for five to 10 minutes. Under the hood, replace the tripsin A DTA with one milliliter of deactivation. Medium gently swell to mix, and then remove the deactivation medium, but leave enough medium behind so dissociated cells are not discarded.

Next, wash the tissues with complete medium twice without losing the cells. Now add one milliliter of complete medium. Then triturate the tissue with the fire polished pipette without forming bubbles until a single cell suspension is obtained.

About eight to 10 passes should suffice. After the duration, centrifuge the cells of 400 Gs for five minutes. Remove the medium and resuspend the cells in one milliliter of complete medium.

Pipette the cells up to four times to achieve a suspension. Begin by counting and assessing the health of the cells in the suspension. Using triam blue exclusion with a hemo cytometer.

Now adjust the cell suspension to 1500 cells per microliter. Then transfer the sterilized micro centrifuge tube caps to 100 millimeter dishes. Place the prepared cover slips onto the caps using forceps.

Do not wash the cover slips and try not to let them dry out. Load 100 microliters of suspension onto each cover slip. This somewhat unusual method provides 90%culture viability and is a critical step to success.

Then close the Petri dish and transfer it to the incubator for an hour after the incubation, carefully transfer the cover slips with the medium to 24 well plates containing 400 microliters of complete medium warm to 37 degrees Celsius in each well then incubate the cells overnight. The first few hours are the most critical to cell survival within 24 hours. Gently add half a milliliter of complete medium to each well.

Every two weeks. Change half the media. If the culture turns yellow, then the half media change can be performed sooner, but the change should still be infrequent.

Dopaminergic neurons will survive for more than six weeks under these conditions. After two weeks of culture, immunohistochemistry against tyrosine, hydroxylase, and neuronal markers show that 1%of the cells are dopaminergic. Neuronal projections appear within two hours of plating.

By the end of the first day in culture, axons and dendrites are distinguishable. Ultimately, the neurons survive for more than six weeks and show extensive outgrowth Following this procedure. Other methods like viral transduction, immune fluorescence or electrophysiology can be performed in order to answer mechanistic questions in dopaminergic neurons.