The overall goal of this procedure is to reliably identify living dopaminergic neurons in midbrain cultures using tyrosine hydroxlase driven eGFP expression, single cell harvesting, and RNA sequencing library generation. This method can help answer key questions in the Parkinson's disease and drug addiction fields, because it makes it possible to perform gene expression and electrophysiological assays on identified living dopaminergic neurons in culture. The advantage of this technique is that it provides a means to reliably identify living rather than fixed dopaminergic neurons in primary ventral mesencephalic cultures.
Demonstrating the first part of the procedure will be Charlene Kim, a technician from Henry Lester's laboratory. Begin by stabilizing the head of a mouse embryo with forceps firmly placed on the snout, so that the dorsal surface is accessible. Using forceps held in the other hand pinch the layer of skin and the skull just before the ridge of the mesencephalon and peel back the skin and the skull caudelly along the midline.
Place the forceps around the ridge with one tip between the cortex and mesoncephalon and the other over the cerebellum. Pinch and remove the entire midbrain. Place the midbrain in a Petri dish with cold HBSS under a dissecting microscope.
Under the dissecting microscope, flip the brain segment so that the ventral side is now accessible. There are now four quadrants visible. Remove all of the meninges and vasculature by gently grasping the meninges with forceps and pulling upward away from the brain.
Next, place one tong of forceps into the ventricle approximately at the center of the segment. Then to separate the midbrain, make a pinch dorsally, and then pinch medially on each side. Take the lower two quadrants by making laterally cuts on both sides.
The ventral tegmental area and the substantia nigra pars compacta are in the ventral inferior section, which appears dense. Place the dissected tissue containing both the VTA and SNC in fresh HBSS in a separate area of the Petri dish. After dissecting all the brain segments, use forceps and a number 11 scalpel to quarter each midbrain section into pieces of approximately equal size.
Then, use a wide bore P1000 pipette tip to transfer all of the quartered midbrain segments, to a 15 milliliter conical tube. After allowing the tissue to settle and removing the HBSS add 500 microliters of papain solution. Incubate in a 37 degree Celsius water bath for 15 minutes.
Following the incubation, use a wide bore P1000 tip to transfer only the midbrain segments into a one milliliter aliquot of DNase I solution and allow the segments to settle to the bottom of the tube. Then, transfer only the midbrain segments to a 15 milliliter tube containing one milliliter of cold stop solution. Replace the second rinse with one milliliter of fresh stop solution and pipette up and down seven times with a P1000 pipette tip to triturate the cells.
Then underlay the cell suspension by slowly pipetting 200 microliters of 4%BSA solution into the bottom of the tube. After centrifugation at 280 x g for six minutes, aspirate the supernatant with a P1000 and resuspend the cells in one milliliter of plating medium. Perform a cell count using hemocytometer and then dilute the cell suspension to 1000 cells per microliter with plating medium.
Plate 120 microliters of the cell suspension onto poly-D-lysine, poly-L-ornithine, and laminin coated culture dishes immediately after aspiration of laminin, to ensure that the coating does not dry and form an uneven surface. Then, after one hour of incubation, carefully add three milliliters of culture medium to an unseeded area of the culture dish to minimize disruption of cells. After three weeks of culture, rinse the culture dish with two milliliters RNase-free Dulbecco's PBS.
Remove the DPBS. Then replace with one milliliter of fresh DPBS for harvesting. Use the GFP filter set and 40X objective on an inverted epifluoresence microscope to identify fluorescent TH positive neurons in the cultures.
Use the micromanipulator to the pipette over the cell soma. Then use plastic tubing attached to the side port of the micropipette holder to apply gentle suction in order to aspirate the neuron into the glass micropipette. Immediately remove the micropipette containing the cell from the bathing solution and place the tip inside a 0.2 millilter PCR tube collection containing reaction buffer.
Break the tip against the side of the tube near the bottom. Then place a sterile 21 gauge syringe needle in the back of the micorpipette and apply pressure to expel the remaining fluid from the broken tip of the micropipette. Centrifuge the collection tube for five seconds using a desktop microcentrifuge and then freeze it in dry ice.
While working in the PCR cabinet with reagents on ice, or on a chiller block, prepare the first strain master mix plus 10%additional volume at room temperature. And one microliter of three prime primer one and one microliter of quantified RNA Spikes to the sample. Then incubate the sample in the thermocycler for three minutes at 72 degrees Celsius and then put the samples directly onto the PCR cooler rack.
Next, add 5.5 microliters of the prepared master mix to the reaction tube in a PCR cooler rack and mix by pipetting gently. After centrifuging the tube for five seconds incubate in a thermocycler set to the parameters now shown on screen. To purify the first-strand cDNA add 25 microliters of vortexed room temperature magnetic beads to the sample.
Mix by pipetting the entire volume up and down at least 10 times. Then incubate at room temperature for eight minutes to allow the cDNA to bind to the beads. Then place the samples and magnetic beads on the magnetic separation device until the liquid appears completely clear and there are no beads left in the supernatant.
Aspirate and discard the supernatant. Spin the sample for five seconds to collect the liquid from the side of the tube. Place the samples on the magnetic separation device for 30 seconds.
Then remove all the remaining supernatant with the P10 pipetter to leave just the beads with bound DNA. Add 50 microliters of the ds-cDNA PCR master mix and then run the second strand synthesis PCR program to obtain the double stranded cDNA. This histogram shows the number of protein coding genes detected in single cell dopaminergic RNA sequencing libraries generated from TH-eGFP positive cells in fragments per kilobase of transcript per million mapped reads.
6, 000 to 8, 000 protein genes were detected in the single cell libraries. Once mastered this technique can be done in four to five weeks, including time to derive the cells, allow for the maturation of the cells in culture, the cell harvesting and the library generation steps, the sequencing of the libraries, and the preliminary analysis. While attempting this procedure, it's important to remember to keep an RNAse-free workspace for the RNA seek library generation and cell harvesting, to maintain sterile techniques through the cell culturing and to make pipettes with appropriately sized diameter for the single cell harvesting.
Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as using a fume hood, avoiding skin and eye contact, keeping away from heat and open flames, and wearing the appropriate personal protective clothing should always be taken. Following this procedure, other methods like gene expression and gene network analysis can be performed in order to answer additional questions such as how drug treatments effect protein expression in dopaminergic cells.
